Anti-viral compounds

ABSTRACT

Disclosed herein are compounds and related compositions for the treatment of viral infection, including RNA viral infection, and compounds that can modulate the RIG-I pathway in vertebrate cells, including compounds that can activate the RIG-I pathway.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/752,846 filed Jan. 15, 2013, the entire contents of whichare incorporated by reference herein.

FIELD OF THE DISCLOSURE

Compounds and methods disclosed herein are useful for treating viralinfection in vertebrates, including RNA viral infections.

BACKGROUND OF THE DISCLOSURE

As a group, RNA viruses represent an enormous public health problem inthe U.S. and worldwide. Well-known RNA viruses include influenza virus(including the avian and swine isolates), hepatitis C virus (HCV), WestNile virus, SARS-coronavirus, respiratory syncytial virus (RSV), andhuman immunodeficiency virus (HIV).

More than 170 million people worldwide are infected by HCV, and 130million of those are chronic carriers at risk of developing chronicliver diseases (cirrhosis, carcinoma, and liver failure). As such, HCVis responsible for two thirds of all liver transplants in the developedworld. Recent studies show that the death rate from HCV infection isrising due to the increasing age of chronically infected patients.Likewise seasonal flu infects 5-20% of the population resulting in200,000 hospitalizations and 36,000 deaths each year.

Compared to influenza and HCV, West Nile virus causes the lowest numberof infections, 981 in the United States in 2010. Twenty percent ofinfected patients develop a severe form of the disease, resulting in a4.5% mortality rate. Unlike influenza and HCV, there are no approvedtherapies for the treatment of West Nile virus infection, and it is ahigh-priority pathogen for drug development due to its potential as abioterrorist agent.

Among the RNA viruses listed, vaccines exist only for influenza virus.Accordingly, drug therapy is essential to mitigate the significantmorbidity and mortality associated with these viruses. Unfortunately,the number of antiviral drugs is limited, many are poorly effective, andnearly all are plagued by the rapid evolution of viral resistance and alimited spectrum of action. Moreover, treatments for acute influenza andHCV infections are only moderately effective. The standard of care forHCV infection, PEGylated interferon and ribavirin, is effective in only50% of patients, and there are a number of dose-limiting side effectsassociated with the combined therapy. Both classes of acute influenzaantivirals, adamantanes and neuraminidase inhibitors, are only effectivewithin the first 48 hours after infection, thereby limiting the windowof opportunity for treatment. High resistance to adamantanes alreadyrestricts their use, and massive stockpiling of neuraminidase inhibitorswill eventually lead to overuse and the emergence of resistant strainsof influenza.

Most drug development efforts against these viruses target viralproteins. This is a large part of the reason that current drugs arenarrow in spectrum and subject to the emergence of viral resistance.Most RNA viruses have small genomes and many encode less than a dozenproteins. Viral targets are therefore limited. Based on the foregoing,there is an immense and unmet need for effective treatments againstviral infections.

SUMMARY OF THE DISCLOSURE

The compounds and methods disclosed herein shift the focus of viral drugdevelopment away from the targeting of viral proteins to the developmentof drugs that target and enhance the host's innate antiviral response.Such compounds and methods are likely to be more effective, lesssusceptible to the emergence of viral resistance, cause fewer sideeffects and be effective against a range of different viruses.

The RIG-I pathway is intimately involved in regulating the innate immuneresponse to RNA virus infections. RIG-I agonists are expected to beuseful for the treatment of many viruses including, without limitation,HCV, influenza, and West Nile virus. Accordingly, the present disclosurerelates to compounds and methods for treating viral infection, includinginfection by RNA viruses, wherein the compounds can modulate the RIG-Ipathway.

One embodiment of the present disclosure includes a compound representedby the formula

wherein a dashed line indicates the presence or absence of a pi bond; Aand B are each independently a covalent single bond or covalent doublebond linking the L group to the ring and R¹, respectively, L may be alinker group having a structure A-C(═R^(x))—NR⁵—B, A-SO₂—NR⁵—B,A-NR⁵—SO₂—B, A-CH(CF₃)—NR⁵—B, A-NR⁵—CH(CF₃)—B,

A-NR⁵—C(═R^(y))—NR⁵—B, A-CR²R³—R^(x)—B, A-O—CR²R³—B, A-S—CR²R³—B,A-C(R²)═C(R³)—B,

m and n may independently be an integer from 0-5 such that m+n≧1; R¹ maybe R^(a), OR² or NR²R³; each R^(a) may independently be H, optionallysubstituted hydrocarbyl, optionally substituted aryl, or optionallysubstituted heteroaryl; R² and R³ may each independently be R^(a),COR^(a), C(═O)OR^(a), or SO₂R^(a); Y¹, Y², Y³, and Y⁴, may eachindependently be CR⁴ or N; Y⁵, Y⁶, Y⁷, and Y⁸ may each independently beCR⁴, N, or R^(x); each R⁴ may independently be R², OR^(a), NR²R³,SR^(a), SOR^(a), SO₂R^(a), SO₂NHR^(a), N(R⁵)COR^(a), halogen,trihalomethyl, CN, S═O, or nitro; R⁵ may be R^(a), COR^(a), SO₂R^(a), oris not present; W and X may each independently be N, NR^(a), O, S, CR²R⁴or CR⁴; each R^(x) may independently be O, S, CR²R³, or NR⁵; R^(y) maybe S, N—CN, or CHR⁴; and Z¹ and Z² may each independently be C, CR², orN.

Additional embodiments include a compound represented by the formula

wherein R¹⁰, R¹³, R¹⁴, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, and R²² areindependently R^(b), OR^(b), SR^(b), COR^(b), CO₂R^(b), OCOR^(b),NR^(b)R^(c), CON^(b)R^(c), NR^(b)COR^(c), SO₂NR^(b)R^(c), CF₃, CN, NO₂,F, Cl, Br, I, or C₂₋₅ heterocyclyl; each R^(b) is independently H orC₁₋₃ hydrocarbyl, and each R^(c) is independently H or C₁₋₃ alkyl.

Some embodiments of the present disclosure include compounds representedby the formula:

Certain embodiments of the present disclosure include compoundsrepresented by the formula:

wherein R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁶, R¹⁷, and R¹⁸ are independentlyR^(b), OR^(b), COR^(b), CO₂R^(b), OCOR^(b), NR^(b)R^(c), CF₃, CN, NO₂,F, Cl, Br, or I, wherein R^(b) and R^(c) are independently H or C₁₋₃alkyl; and, R⁵ is H or C₁₋₃ alkyl.

Further embodiments of the present disclosure include a compoundrepresented by the formula:

Some embodiments of the present disclosure include a pharmaceuticalcomposition comprising any of the compounds as described herein.

Some embodiments of the present disclosure include methods of treatingor preventing a viral infection in a vertebrate comprising administeringto the vertebrate a pharmaceutical composition as described herein. Insome embodiments, the viral infection is caused by a virus from one ormore of the following families: Arenaviridae, Astroviridae,Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae,Closteroviridae, Comoviridae, Cystoviridae, Flaviviridae, Flexiviridae,Hepevirus, Leviviridae, Luteoviridae, Mononegavirales, Mosaic Viruses,Nidovirales, Nodaviridae, Orthomyxoviridae, Picobirnavirus,Picornaviridae, Potyviridae, Reoviridae, Retroviridae, Sequiviridae,Tenuivirus, Togaviridae, Tombusviridae, Totiviridae, Tymoviridae,Hepadnaviridae, Herpesviridae, Paramyxoviridae or Papillomaviridae. Insome embodiments, the viral infection is influenza virus, Hepatitis Cvirus, West Nile virus, SARS-coronavirus, poliovirus, measles virus,Dengue virus, yellow fever virus, tick-borne encephalitis virus,Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valleyvirus, Powassan virus, Rocio virus, louping-ill virus, Banzi virus,Ilheus virus, Kokobera virus, Kunjin virus, Alfuy virus, bovine diarrheavirus, Kyasanur forest disease virus, respiratory syncytial virus orHIV.

Some embodiments of the methods of the present disclosure includeadministering any of the pharmaceutical compositions described herein asan adjuvant for a prophylactic or therapeutic vaccine. In someembodiments, the method includes vaccinating a vertebrate byadditionally administering a vaccine against influenza virus, HepatitisC virus, West Nile virus, SARS-coronavirus, poliovirus, measles virus,Dengue virus, yellow fever virus, tick-borne encephalitis virus,Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valleyvirus, Powassan virus, Rocio virus, louping-ill virus, Banzi virus,Ilheus virus, Kokobera virus, Kunjin virus, Alfuy virus, bovine diarrheavirus, Kyasanur forest disease virus or HIV.

Some embodiments of the present disclosure include methods of modulatingthe innate immune response in a eukaryotic cell, comprisingadministering to the cell any of the compounds as described herein. Insome embodiments the cell is in vivo. In other embodiments the cell isin vitro.

BRIEF DESCRIPTION OF THE DRAWINGS

The various embodiments of the present disclosure may be betterunderstood when read in conjunction with the following figures, wherein:

FIGS. 1A-1E shows validation and characterization of compound KIN1000(“RLU”=relative luciferase units). Initial “hit” compounds werevalidated by demonstrating dose-dependent induction of theIFNβ-luciferase (IFNβ-LUC, FIG. 1A), ISG56-luciferase (ISG56-LUC, FIG.1D), and the ISG54-luciferase (ISG54-LUC, FIG. 1E) reporter genes. FIG.1B confirms the specificity of KIN1000, which does not induce thenon-specific β-actin promoter (“0.5% DMSO”=vehicle control; “10 μMKIN1000”=β-actinduciferase reporter in presence of KIN1000; “10 μMCompound X”=positive control β-actin induction). In FIG. 1C, the MTSassay demonstrated that KIN1000 did not show evident cytotoxicity tohuman cells treated for 48 hours with the compound. The O.D. value thatrepresents 50% cell mortality is shown by a horizontal line, alsodemonstrating that the CC50 of KIN1000 is greater than 20 μM.

FIGS. 2A-2B shows activation of transcription factors by KIN1000. InFIG. 2A, HeLa cells treated with increasing amounts of KIN1000 showeddose-dependent increase in IRF-3 translocation to the nucleus,quantified by nuclear intensity minus cytoplasmic intensity (“normalizednuclear intensity”). In FIG. 2B, HeLa cells treated with increasingamounts of KIN1000 showed dose-dependent increase in NFκB translocation,quantified by nuclear intensity minus cytoplasmic intensity. “SeV”refers to Sendai virus infection, the positive control.

FIG. 3 shows anti-viral activity of KIN1000. MRC5 cells treated withincreasing amounts of KIN1000 showed dose-dependent decrease ininfection by influenza virus.

FIGS. 4A-4D shows Luminex® (Luminex Corp., Austin Tex.) quantifiedlevels of cytokine expression induced by KIN1000. Human dendritic cellstreated with increasing amounts of KIN1000 showed dose-dependentexpression of cytokines including IL-8 (FIG. 4A), MCP-1 (CCL2) (FIG.4B), and MIP-1α and β (CCL3 (FIG. 4C) and CCL4 (FIG. 4D), respectively).

FIGS. 5A-5E shows induction of gene expression by KIN1000 and itsderivative compound KIN1148. FIG. 5A shows gene expression levels ofIFIT2 (FIG. 5A) and OAS1 (FIG. 5B) in HeLa cells over time from 4-24hours post treatment with 10 uM KIN1000 (grey) or KIN1148 (black). FIG.5C shows gene expression levels of IFIT2 in PH5CH8 cells (left) treatedwith KIN1000 (solid grey bars) or KIN1148 (solid black bars), and inHeLa cells (right) treated with KIN1000 (grey striped bars) or KIN1148(black checked bars). In each test group, the three vertical barsrepresent 5, 10, and 20 μM compound (KIN1000 or KIN1148), respectivelyGene expression levels of IFIT2 (FIG. 5D), OAS1 (FIG. 5E), and MxA (FIG.5F) in primary HUVEC cells that were treated with 1 μM KIN1000 (grey) or1 μM KIN1148 (black).

FIGS. 6A-6B shows antiviral activity of KIN1000 and KIN1148 againstrespiratory syncytial virus. FIG. 6A shows that HeLa cells treated withincreasing amount of KIN1000 and KIN1148 showed dose-dependent decreasein infection by RSV. FIG. 6B shows that KIN1148 showed antiviralactivity against RSV when drug is added up to 24 hours prior toinfection.

FIGS. 7A-7B shows antiviral activity of KIN1148 against Influenza Avirus Udorn/72. H292 cells (FIG. 7A) and HEK293 cells (FIG. 7B) treatedwith 2 μM (H292) or 10 μM (HEK293) of KIN1148 showed decrease ininfection by virus.

FIG. 8 shows antiviral activity of KIN1148 against Dengue virus type 2.Huh 7 cells treated with increasing amounts of KIN1148 showeddose-dependent decrease in infection by virus.

FIG. 9 shows antiviral activity of KIN1148 against Hepatitis B virus.HepAD38 cells treated with increasing amounts of KIN1148 showeddose-dependent decrease in supernatant levels of virus. The O.D. valuethat represents no HBV in the supernatant is shown by a horizontal linelabeled “NO HBV CELLS.”

FIG. 10 shows IgG antibody production induced by KIN1000 and KIN1148 invivo. Animals (Lewis female rats, 10-12 weeks old) were vaccinated withsuspensions of OVA in PBS, OVA+polyl:C, OVA+KIN1000 or OVA+KIN1148subcutaneously in the footpad and base of tail (0.025 mL injectionvolume per site). Animals were boosted identically at 2 and 8 weeks postpriming. Animals were bled at the indicated time points, sera wasprepared and antibody levels were detected by ELISA. OD450 values forvaccine preparations containing KIN1000 (large checked bars) and KIN1148(horizontal striped bars) were normalized to values obtained fromanimals that received OVA in PBS alone as vaccines. Poly I:C (smallchecked bars) was used as a control adjuvant.

FIG. 11 shows cellular response elicited by KIN compound vaccination.Delatyed type hypersensitivity responses elicited 2 weeks after thefirst boost (4 weeks post prime) were measured. Animals were challengedby injection of 0.02 mL of PBS (left ear pinna) or 0.02 mL of OVA (1mg/mL) in PBS (right ear pinna) at indicated time point. 24 hours laterear thickness was measured with calipers. The calculated differencebetween right ear and left ear is shown. “OVA+K1148” (vertical stripedbar)=difference in ear thickness in animal injected with vaccinecontaining KIN1148. Poly I:C (“OVA+pl:C;” horizontal striped bar) wasused as a control adjuvant.

DETAILED DESCRIPTION

The present disclosure provides compounds and methods that shift thefocus of viral treatments away from the targeting of viral proteins tothe development of drugs that target and enhance the host (patient's)innate antiviral response. Such compounds and methods are likely to bemore effective, less susceptible to the emergence of viral resistance,cause fewer side effects and be effective against a range of differentviruses.

The RIG-I pathway is intimately involved in regulating the innate immuneresponse to RNA virus infections. RIG-I is a cytosolic pathogenrecognition receptor that is essential for triggering immunity to a widerange of RNA viruses. RIG-I is a double-stranded RNA helicase that bindsto motifs within the RNA virus genome characterized by homopolymericstretches of uridine or polymeric U/A motifs. Binding to RNA induces aconformation change that relieves RIG-I signaling repression by anautologous repressor domain, thus allowing RIG-I to signal downstreamthrough its tandem caspase activation and recruitment domains (CARDs).RIG-I signaling is dependent upon its NTPase activity, but does notrequire the helicase domain. RIG-I signaling is silent in resting cells,and the repressor domain serves as the on-off switch that governssignaling in response to virus infection.

RIG-I signaling is transduced through IPS-1 (also known as Cardif, MAVs,and VISA), an essential adaptor protein that resides in the outermitochondrial membrane. IPS-1 recruits a macromolecular signalingcomplex that stimulates the downstream activation of IRF-3, atranscription factor that induces the expression of type I IFNs andvirus-responsive genes that control infection. Compounds that triggerRIG-I signaling directly or through modulation of RIG-I pathwaycomponents, including IRF-3, present attractive therapeutic applicationsas antivirals or immune modulators.

A high-throughput screening approach was used to identify compounds thatmodulate the RIG-I pathway, a key regulator of the cellular innateimmune response to RNA virus infection. In particular embodiments,validated RIG-I agonist lead compounds were demonstrated to specificallyactivate interferon regulatory factor-3 (IRF-3). In additionalembodiments they exhibit one or more of the following: they induce theexpression of interferon-stimulated genes (ISGs), have low cytotoxicityin cell-based assays, are suitable for analog development and SARstudies, have drug-like physiochemical properties, and have antiviralactivity against influenza A virus and/or HCV.

As discussed below, these compounds represent a new class of potentialantiviral therapeutics. Although the disclosure is not bound by aspecific mechanism of action of the compounds in vivo, the compounds areselected for their modulation of the RIG-I pathway. In certainembodiments, the modulation is activation of the RIG-I pathway.Compounds and methods disclosed herein function to, one or more of,decrease viral protein, viral RNA, and infectious virus in cell culturemodels of HCV and/or influenza virus.

Examples of antiviral compounds and pharmaceutical formulations preparedtherefrom are described in detail in U.S. Provisional Application Ser.No. 61/542,049, filed Sep. 30, 2011 and PCT International ApplicationNo. PCT/US2012/057646, filed Sep. 27, 2012, the disclosures of each ofwhich are incorporated herein in their entirety by this reference. Forexample, for one embodiment, the disclosure herein relates to a class ofcompounds of represented by the following formula:

wherein a dashed line indicates the presence or absence of a pi bond; R¹may be R^(a), OR² or NR²R³; each R^(a) may independently beindependently H, optionally substituted hydrocarbyl, optionallysubstituted aryl, or optionally substituted heteroaryl; R² and R³ mayeach independently be R^(a), COR^(a), C(═O)OR^(a), or SO₂R^(a); Y¹, Y²,Y³ and Y⁴ may each independently be CR⁴ or N; each R⁴ may independentlybe R², OR^(a), NR²R³, SR^(a), SOR^(a), SO₂R^(a), SO₂NHR^(a),N(R⁵)COR^(a), halogen, trihalomethyl, CN, S═O, or nitro; R⁵ may beR^(a), COR^(a), SO₂R^(a), or is not present; V may be CR², CR²R³, C═O,COCR²R³, or C═NR²; and, W and X may each independently be N, NR^(a), O,S, CR²R⁴ or CR⁴.

According to certain embodiments, effective antiviral compounds havingFormula 1 may have an amide linker between the ring structure of Formula1 and the group R¹. According to these embodiments, V may comprise C═Oand R⁵ may be H. However, the central amide linker present in thesetypes of structures may be susceptible to protease hydrolysis, whichmight diminish the efficacy of the adjuvant. Alternative linking groups,such as, but no limited to amide isosteres or other small stable linkingstructures, may display lower levels of hydrolysis and in certainembodiments be less susceptible to hydrolysis by proteases. For example,in certain embodiments of the present disclosure, compounds having astructure according to Formula 1, except where the amide is replacedwith an amide isostere linkers such as methylene ethers (—CH₂O—) andmethylene amines (—CH₂NH—) may retain the IRF3 activity of the moleculewhile being more stable to hydrolysis. In addition, embodiments of theantiviral compounds represented by Formula 1 possess scaffold structuresof a modular nature which should be amenable to preparation of analogshaving different linking structures.

According to certain embodiments, the present disclosure is directed tocompounds having a structure represented by the formula:

where R¹ may be R^(a), OR² or NR²R³; each R^(a) may independently beindependently H, optionally substituted hydrocarbyl, optionallysubstituted aryl, or optionally substituted heteroaryl; R² and R³ mayeach independently be R^(a), COR^(a), C(═O)OR^(a), or SO₂R^(a); Y¹, Y²,Y³ and Y⁴ may each independently be CR⁴ or N; each R⁴ may independentlybe R², OR^(a), NR²R³, SR^(a), SOR^(a), SO₂R^(a), SO₂NHR^(a),N(R⁵)COR^(a), halogen, trihalomethyl, CN, S═O, or nitro; R⁵ may beR^(a), COR^(a), C(═O)OR^(a), SO₂R^(a), or is not present; and, W and Xmay each independently be N, NR^(a), O, S, CR²R⁴ or CR⁴.

According to any of the structural formulas herein, a dashed lineindicates the presence or absence of a pi bond between the indicatedatoms in the structure. That is, when two atoms in the structure areconnected by a solid line and a dashed line, the atoms may be connectedby a covalent singe bond (i.e., a sigma bond, when the dashed linerepresents the absence of a pi bond), a covalent double bond (i.e., asigma and pi bond, when the dashed line represents the presence of a pibond and the bonds are not part of an aromatic structure), or adelocalized “double bond” (i.e., a sigma bond and a pi bond that is partof a delocalized aromatic structure or other delocalized system). Thenumber of bonds on any atom in any structural formula will be limited bythe maximum valence of the atom; and may include the valence of an atomas determined by ionic charge. According to various formulas representedherein, A and B may each independently represent a single bond or doublecovalent bond between the two structural features connected by A or B.For example, a substructure shown as C¹-A-C² or C¹-B—C² may indicateeither C¹—C² (i.e., a covalent single bond between the atoms C¹ and C²)or C¹═C² (i.e., a covalent double between the atoms C¹ and C²).Likewise, when two (or more) structural elements are indicated asseparate elements, for example, C¹-A and A-C² or C¹—B and B—C², the A orB indicates the presence of a bond between the two structural elements,such that the over all structure may be represented by C¹—C², where A orB indicated the covalent bond between the two elements. For example,structural elements represented by R^(a)-A, A-L-B and B—R^(b) may betaken to indicate the overall structure R^(a)-L-R^(b), where the threestructural elements are connected by covalent bonds A and B (asindicated by the dash). In certain embodiments, A or B may represent adelocalized double bond. In embodiments where A or B are between an atomof a structure and a linking group (i.e., a grouping of two or moreatoms that link two or more substructures within a compound), such asC-A-L, or L-B—R¹, where L has a generic structure shown as A-LINKER-B(where “LINKER” represents the atom structure of the linking group “L”),the A or B group indicates a single or double covalent bond (ordelocalized double bond) as represented by C-LINKER-B, C=LINKER-B,C-LINKER=B, or C=LINKER=B, where the single or double bond is attachedto the atoms in LINKER that has the A and/or B attached thereto.

According to various embodiments of the antiviral compounds describedherein, the group L may be a linker having a structure represented by:A-C(═R^(x))—NR⁵—B, A-SO₂—NR⁵—B, A-NR⁵—SO₂—B, A-CH(CF₃)—NR⁵—B,A-NR⁵—CH(CF₃)—B,

A-NR⁵—C(═R^(y))—NR⁵—B, A-CR²R³—R^(x)—B, A-O—CR²R³—B, A-S—CR²R³—B,A-C(R²)═C(R³)—B,

where m and n may each independently be an integer from 0 to 5 and areselected such that m+n≦1, Y⁵, Y⁶, Y⁷ and Y⁸ may each independently beCR⁴, N, or R^(x); each R^(x) may independently be O, S, CR²R³, or NR⁵;R^(y) may be S, N—CN, or CHR⁴; and Z¹ and Z² may each independently beO, CR², or N.

According to one embodiment, the group L may have a structure of anamide, thioamide, enamine, or amidine where the sp² carbon forms a bondwith the ring carbon of Formula I (via A) and the nitrogen atom forms abond with the R¹ group (via B). According to these embodiments, L may bea linker having a structure A-C(═R^(x))—NR⁵—B, where R^(x) may be O(amide), S (thioamide), CR²R³ (enamine), or NR⁵ (amidine); and R², R³,and R⁵ are as defined herein.

According to other embodiments, the group L may have a structure of asulfonamide where the sulfur atom forms a bond with the ring carbon ofFormula I (via A) and the nitrogen atom forms a bond with the R¹ group(via B) or alternatively, the nitrogen atom forms a bond with the ringcarbon of Formula I (via A) and the sulfur atom forms a bond with the R¹group (via B). According to these embodiments, L may be a sulfonamidelinker having a structure A-SO₂—NR⁵—B or A-NR⁵—SO₂—B, where R⁵ is asdefined herein.

According to other embodiments, the group L may have a structure of a2,2,2-trifluoroethylamine where the C¹ carbon atom of the2,2,2-trifluoroethyl group forms a bond with the ring carbon of FormulaI (via A) and the nitrogen atom forms a bond with the R¹ group (via B)or alternatively, the nitrogen atom forms a bond with the ring carbon ofFormula I (via A) and the C¹ carbon atom of the 2,2,2-trifluoroethylgroup forms a bond with the R¹ group (via B). According to theseembodiments, L may be a 2,2,2-trifluoroethylamine linker having astructure A-CH(CF₃)—NR⁵—B or A-NR⁵—CH(CF₃)—B, where R⁵ is as definedherein.

According to other embodiments, the group L may have a structure of a3,3-oxetanylamine where the C² carbon atom of the oxetane forms a bondwith the ring carbon of Formula I (via A) and the nitrogen atom forms abond with the R¹ group (via B) or alternatively, the nitrogen atom formsa bond with the ring carbon of Formula I (via A) and the C² carbon atomof the oxetane forms a bond with the R¹ group (via B). According tothese embodiments, L may be a 3,3-oxetanylamine linker having astructure:

where R⁵ is as defined herein.

According to other embodiments, the group L may have a structure of a1,1-cyclopropylamine where the C¹ carbon atom of the cyclopropane formsa bond with the ring carbon of Formula I (via A) and the nitrogen atomforms a bond with the R¹ group (via B) or alternatively, the nitrogenatom forms a bond with the ring carbon of Formula I (via A) and the C¹carbon atom of the cyclopropane forms a bond with the R¹ group (via B).According to these embodiments, L may be a 1,1-cyclopropylamine linkerhaving a structure:

where R⁵ is as defined herein.

According to other embodiments, the group L may have a structure of a1-fluoro-2-aminoethylene where the C¹ carbon atom of the ethylene formsa bond with the ring carbon of Formula I (via A) and the C² carbon atomof the ethylene forms a bond with the R¹ group (via B) or alternatively,the C¹ carbon atom of the ethylene forms a bond with the ring carbon ofFormula I (via A) and the C¹ carbon atom of the ethylene forms a bondwith the R¹ group (via B). According to these embodiments, L may be a1-fluoro-2-aminoethylene linker having a structure:

According to other embodiments, the group L may have a structure of asaturated, unsaturated or aromatic 5-membered 1,3-carbocyclyl or1,3-heterocyclyl ring where the C¹ carbon atom or N¹ nitrogen atom ofthe 5-membered carbocyclic or heterocyclic ring forms a bond with thering carbon of Formula I (via A) and the C³ carbon atom or N³ nitrogenatom of the 5-membered carbocyclic or heterocyclic ring forms a bondwith the R¹ group (via B) or alternatively, the C³ carbon atom or N³nitrogen atom of the 5-membered carbocyclic or heterocyclic ring forms abond with the ring carbon of Formula I (via A) and the C¹ carbon atom orN¹ nitrogen atom of the 5-membered carbocyclic or heterocyclic ringforms a bond with the R¹ group (via B). In another embodiment, the groupL may be a saturated, unsaturated or aromatic 5-membered 1,2-carbocyclylor 1,2-heterocyclyl ring where the C¹ carbon atom or N¹ nitrogen atom ofthe 5-membered carbocyclic or heterocyclic ring forms a bond with thering carbon of Formula I (via A) and the C² carbon atom or N² nitrogenatom of the 5-membered carbocyclic or heterocyclic ring forms a bondwith the R¹ group (via B) or alternatively, the C² carbon atom or N²nitrogen atom of the 5-membered carbocyclic or heterocyclic ring forms abond with the ring carbon of Formula I (via A) and the C¹ carbon atom orN¹ nitrogen atom of the 5-membered carbocyclic or heterocyclic ringforms a bond with the R¹ group (via B). The 5-membered ring may containcarbon, nitrogen, oxygen and/or sulfur atoms as ring atoms. The5-membered ring may be saturated (i.e., all single bonds), have onedouble bond, have two double bonds, or be aromatic (i.e., a delocalizedpi system containing 6 pi electrons). According to these embodiments, Lmay be a 5-membered carbocyclic or heterocyclic ring linker having astructure:

where Z¹, Z², Y⁵, Y⁶ and Y⁷ are as defined herein. In specificembodiments, the linker may be a carbocyclic ring comprising acyclopentane ring or a cyclopentene ring. In other embodiments, thelinker may be a five membered ring with one or more heteroatoms such asN, O and/or S. In embodiments where the linker is an aromatic 5-memberedring, at least one ring atom is a heteroatom. Non-limiting examples ofaromatic ring structures may include a furan, a thiofuran, a pyrrole, animidazole, a pyrazole, an oxazole, an isoxazole, a thiazole, aisothiazole, an azaoxazole, a triazole, a tetrazole, etc., where theheteroatom(s) may be located at the various positions of the 5-memberedring. Other non-limiting examples of five membered rings may include adihydro- and tetrahydrofurans, dihydro- and tetrahydrothiofurans,pyrrolines, pyrrolidines, imidazolidine, imidazolines, pyrazolidines,pyrazolines, etc., where the heteroatom(s) may be located at the variouspositions of the 5-membered ring. In certain embodiments, where Z¹ andZ² each represent sp³ hybridized carbon atoms, the bonds A and B may beon the same face of the ring (i.e., cis) or on opposite faces of thering (i.e., trans).

In other embodiments, the linker L may have a structure of a saturated,unsaturated or aromatic 5-membered 1,3-carbocyclyl or 1,3-heterocyclylring, as described herein, with an amine substituent bonded to the 1 or3 position of the ring, where the C¹ carbon atom or N¹ nitrogen atom ofthe 5-membered carbocyclic or heterocyclic ring forms a bond with thering carbon of Formula I (via A) and the nitrogen atom of the aminesubstituent forms a bond with the R¹ group (via B) or alternatively, thenitrogen atom of the amine substituent forms a bond with the ring carbonof Formula I (via A) and the C¹ carbon atom or N¹ nitrogen atom of the5-membered carbocyclic or heterocyclic ring forms a bond with the R¹group (via B). According to these embodiments, L may be an aminesubstituted 5-membered carbocyclic or heterocyclic ring linker having astructure:

where R⁵, Z¹, Z², Y⁵, Y⁶ and Y⁷ are as defined herein. In certainembodiments, where Z¹ and Z² each represent sp³ hybridized carbon atoms,the substituent bonds to A and B or the NR⁵ group may be on the sameface of the ring (i.e., cis) or on opposite faces of the ring (i.e.,trans).

In further embodiments, the linker L may have a structure of asaturated, unsaturated or aromatic 5-membered 1,2-carbocyclyl or1,2-heterocyclyl ring, as described herein, with an amine substituentbonded to the 1 or 2 position of the ring, where the C¹ carbon atom orN¹ nitrogen atom of the 5-membered carbocyclic or heterocyclic ringforms a bond with the ring carbon of Formula I (via A) and the nitrogenatom of the amine substituent forms a bond with the R¹ group (via B) oralternatively, the nitrogen atom of the amine substituent forms a bondwith the ring carbon of Formula I (via A) and the C¹ carbon atom or N¹nitrogen atom of the 5-membered carbocyclic or heterocyclic ring forms abond with the R¹ group (via B). According to these embodiments, L may bean amine substituted 5-membered carbocyclic or heterocyclic ring linkerhaving a structure:

where R⁵, Z¹, Z², Y⁵, Y⁶ and Y⁷ are as defined herein. In certainembodiments, where Z¹ and Z² each represent spa hybridized carbon atoms,the substituent bonds to A and B or the NR⁵ group may be on the sameface of the ring (i.e., cis) or on opposite faces of the ring (i.e.,trans).

According to other embodiments, the group L may have a structure of asaturated, unsaturated or aromatic 6-membered 1,3-carbocyclyl or1,3-heterocyclyl ring where the C¹ carbon atom or N¹ nitrogen atom ofthe 6-membered carbocyclic or heterocyclic ring forms a bond with thering carbon of Formula I (via A) and the C³ carbon atom or N³ nitrogenatom of the 6-membered carbocyclic or heterocyclic ring forms a bondwith the R¹ group (via B) or alternatively, the C³ carbon atom or N³nitrogen atom of the 6-membered carbocyclic or heterocyclic ring forms abond with the ring carbon of Formula I (via A) and the C¹ carbon atom orN¹ nitrogen atom of the 6-membered carbocyclic or heterocyclic ringforms a bond with the R¹ group (via B). In another embodiment, the groupL may be a saturated, unsaturated or aromatic 6-membered 1,2-carbocyclylor 1,2-heterocyclyl ring where the C¹ carbon atom or N¹ nitrogen atom ofthe 6-membered carbocyclic or heterocyclic ring forms a bond with thering carbon of Formula I (via A) and the C² carbon atom or N² nitrogenatom of the 6-membered carbocyclic or heterocyclic ring forms a bondwith the R¹ group (via B) or alternatively, the C² carbon atom or N²nitrogen atom of the 6-membered carbocyclic or heterocyclic ring forms abond with the ring carbon of Formula I (via A) and the C¹ carbon atom orN¹ nitrogen atom of the 6-membered carbocyclic or heterocyclic ringforms a bond with the R¹ group (via B). In another embodiment, the groupL may be a saturated, unsaturated or aromatic 6-membered 1,4-carbocyclylor 1,4-heterocyclyl ring where the C¹ carbon atom or N¹ nitrogen atom ofthe 6-membered carbocyclic or heterocyclic ring forms a bond with thering carbon of Formula I (via A) and the C⁴ carbon atom or N⁴ nitrogenatom of the 6-membered carbocyclic or heterocyclic ring forms a bondwith the R¹ group (via B) or alternatively, the C⁴ carbon atom or N⁴nitrogen atom of the 6-membered carbocyclic or heterocyclic ring forms abond with the ring carbon of Formula I (via A) and the C¹ carbon atom orN¹ nitrogen atom of the 6-membered carbocyclic or heterocyclic ringforms a bond with the R¹ group (via B). The 6-membered ring may containcarbon, nitrogen, oxygen and/or sulfur atoms as ring atoms. The6-membered ring may be saturated (i.e., all single bonds), have onedouble bond, have two double bonds, or be aromatic (i.e., a delocalizedpi system containing 6 pi electrons). According to these embodiments, Lmay be a 6-membered carbocyclic or heterocyclic ring linker having astructure:

where Z¹, Z², Y⁵, Y⁶ Y⁷ and Y⁸ are as defined herein. The atoms in thesix membered ring may be substituted or unsubstituted. In specificembodiments, the linker may be a carbocyclic ring comprising acyclohexane ring, a cyclohexene ring or cyclohexadiene ring. In otherembodiments, the linker may be a six membered ring with one or moreheteroatoms such as N, O and/or S. In embodiments where the linker is anaromatic 6-membered ring, the ring may be a phenyl ring (i.e., all ringatoms are carbon) or at least one ring atom may be a heteroatom.Non-limiting examples of aromatic ring structures may include a phenyl,a pyridine, a pyridazine, a pyrimidine, a pyrazine, a triazine, atetraazine, etc., where the heteroatom(s) may be located at the variouspositions of the 6-membered ring. Other non-limiting examples of sixmembered rings may include pyran, dihydro- and tetrahydropyrans,thiopyrans, dihydro- and tetrahydrothiopyrans, piperidines, piperizines,hexahydro-, tetrahydro-, and dihydropyrimidines, morpholines,thiomorpholines, dioxanes, oxothianes, thianes, dithianes, etc., wherethe heteroatom(s) may be located at the various positions of the6-membered ring. In certain embodiments, where Z¹ and Z² each representsp³ hybridized carbon atoms, the bonds A and B may be on the same faceof the ring (i.e., cis) or on opposite faces of the ring (i.e., trans).

In other embodiments, the linker L may have a structure of a saturated,unsaturated or aromatic 6-membered 1,3-carbocyclyl or 1,3-heterocyclylring, as described herein, with an amine substituent bonded to the 1 or3 position of the ring, where the C¹ carbon atom or N¹ nitrogen atom ofthe 6-membered carbocyclic or heterocyclic ring forms a bond with thering carbon of Formula I (via A) and the nitrogen atom of the aminesubstituent forms a bond with the R¹ group (via B) or alternatively, thenitrogen atom of the amine substituent forms a bond with the ring carbonof Formula I (via A) and the C¹ carbon atom or N¹ nitrogen atom of the6-membered carbocyclic or heterocyclic ring forms a bond with the R¹group (via B). According to these embodiments, L may be an aminesubstituted 6-membered carbocyclic or heterocyclic ring linker having astructure:

where R⁵, Z¹, Z², Y⁵, Y⁶, Y⁷, and Y⁸ are as defined herein. In certainembodiments, where Z¹ and Z² each represent sp³ hybridized carbon atoms,the substituent bonds to A and B or the NR⁵ group may be on the sameface of the ring (i.e., cis) or on opposite faces of the ring (i.e.,trans).

In further embodiments, the linker L may have a structure of asaturated, unsaturated or aromatic 6-membered 1,2-carbocyclyl or1,2-heterocyclyl ring, as described herein, with an amine substituentbonded to the 1 or 2 position of the ring, where the C¹ carbon atom orN¹ nitrogen atom of the 6-membered carbocyclic or heterocyclic ringforms a bond with the ring carbon of Formula I (via A) and the nitrogenatom of the amine substituent forms a bond with the R¹ group (via B) oralternatively, the nitrogen atom of the amine substituent forms a bondwith the ring carbon of Formula I (via A) and the C¹ carbon atom or N¹nitrogen atom of the 6-membered carbocyclic or heterocyclic ring forms abond with the R¹ group (via B). According to these embodiments, L may bean amine substituted 6-membered carbocyclic or heterocyclic ring linkerhaving a structure:

where R⁵, Z¹, Z², Y⁵, Y⁶, Y⁷ and Y⁸ are as defined herein. In certainembodiments, where Z¹ and Z² each represent spa hybridized carbon atoms,the substituent bonds to A and B or the NR⁵ group may be on the sameface of the ring (i.e., cis) or on opposite faces of the ring (i.e.,trans).

In further embodiments, the linker L may have a structure of asaturated, unsaturated or aromatic 6-membered 1,4-carbocyclyl or1,4-heterocyclyl ring, as described herein, with an amine substituentbonded to the 1 or 4 position of the ring, where the C¹ carbon atom orN¹ nitrogen atom of the 6-membered carbocyclic or heterocyclic ringforms a bond with the ring carbon of Formula I (via A) and the nitrogenatom of the amine substituent at the 4-position forms a bond with the R¹group (via B) or alternatively, the nitrogen atom of the aminesubstituent at the 4-position forms a bond with the ring carbon ofFormula I (via A) and the C¹ carbon atom or N¹ nitrogen atom of the6-membered carbocyclic or heterocyclic ring forms a bond with the R¹group (via B). According to these embodiments, L may be an aminesubstituted 6-membered carbocyclic or heterocyclic ring linker having astructure:

where R⁵, Z¹, Z², Y⁵, Y⁶, Y⁷ and Y⁸ are as defined herein. In certainembodiments, where Z¹ and Z² each represent spa hybridized carbon atoms,the substituent bonds to A and B or the NR⁵ group may be on the sameface of the ring (i.e., cis) or on opposite faces of the ring (i.e.,trans).

In further embodiments, the linker L may have a structure of a5-membered 3,4-diamino substituted 1,1-dioxo thio-2,5-imidazole ring,with amine substituents bonded to the 3 or 4 positions of the ring,where the nitrogen atom of the amine substituent at the 3 position ofthe ring forms a bond with the ring carbon of Formula I (via A) and thenitrogen atom of the amine substituent at the 4 position of the ringforms a bond with the R¹ group (via B). According to these embodiments,L may be a 3,4-diamino substituted 1,1-dioxo thio-2,5-imidazole ringlinker having a structure:

where R⁵ is as defined herein.

In further embodiments, the linker L may have a structure of a thiourea,a N-cyanoguanidine, or a 1,1-diaminoalkene, where the nitrogen atom ofone of the amino groups forms a bond with the ring carbon of Formula I(via A) and the nitrogen atom of the other amino group forms a bond withthe R¹ group (via B). According to these embodiments, L may be athiourea, a N-cyanoguanidine, or a 1,1-diaminoalkene linker having astructure: A-NR⁵—C(═R^(y))—NR⁵—B, where R^(y) may be S, N—CN, or CHR⁴,and R⁴ and R⁵ are as defined herein.

In other embodiments, the linker L may have a structure having a linkingchain of two atoms between the ring carbon of Formula I and R1, whereone of the atoms is a substituted or unsubstituted carbon atom and theother atom may be a substituted or unsubstituted carbon or nitrogen atomor a sulfur or oxygen atom (i.e. a two carbon alkyl linker, an aminelinker, an ether linker or a thioether linker), where the carbon atomforms a bond with the ring carbon of Formula I (via A) and the othercarbon atom, nitrogen atom, oxygen atom or sulfur atom of the linkerforms a bond with the R¹ group (via B), or alternatively, the othercarbon atom, nitrogen atom, oxygen atom or sulfur atom of the linkerforms a bond with the ring carbon of Formula I (via A) and the carbonatom forms a bond with the R¹ group (via B). According to theseembodiments, L may be a two carbon alkyl linker, an amine linker, anether linker or a thioether linker having a structure: A-CR²R³—R^(x)—B,A-O—CR²R³—B, or A-S—CR²R³—B, where R^(x), R² and R³ are as definedherein.

In other embodiments, the linker L may have a structure of a di-, mono-or unsubstituted ethylene unit (i.e., two carbon unit connected by adouble bond), where one carbon atom of the ethylene group forms a bondwith the ring carbon of Formula I (via A) and the other carbon atom ofthe ethylene group forms a bond with the R¹ group (via B). According tothese embodiments, L may be a two carbon ethylene linker having astructure: A-C(R²)αC(R³)—B, where R² and R³ are as defined herein andthe bonds A and B may be on the same side of the double bond (i.e., cis)or on opposite sides of the double bond (i.e., trans).

In other embodiments, the linker L may comprise a 1,2-cyclopropane,1,2-epoxide, 1,2-thioepoxide, or 1,2-aziridine, where one carbon atom ofthe ring forms a bond with the ring carbon of Formula I (via A) andanother carbon atom of the ring forms a bond with the R¹ group (via B).According to these embodiments, L may be a 1,2-cyclopropane,1,2-epoxide, 1,2-thioepoxide, or 1,2-aziridine linker having astructure:

where R^(x) may be as defined herein and the bonds A and B may be on thesame face of the ring (i.e., cis) or on opposite faces of the ring(i.e., trans).

In still other embodiments, the linker L may comprise a 1,2-cyclobutane,1,2-oxetane, 1,2-thiooxetane, or 1,2-azetidine, where one carbon atom ofthe ring forms a bond with the ring carbon of Formula I (via A) andanother carbon atom of the ring forms a bond with the R¹ group (via B).According to these embodiments, L may be a 1,2-cyclobutane, 1,2-oxetane,1,2-thiooxetane, or 1,2-azetidine linker having a structure:

where each R^(x) may independently be O, S, CR²CR³, or NR⁵, and thebonds A and B may be on the same face of the ring (i.e., cis) or onopposite faces of the ring (i.e., trans).

In still other embodiments, the linker L may comprise an aminosubstituted-1,2-cyclobutane, amino substituted-1,2-oxetane, aminosubstituted-1,2-thiooxetane, or amino substituted-1,2-azetidine, wherethe amino substituent on the C¹ carbon atom of the ring forms a bondwith the ring carbon of Formula I (via A) and C² carbon atom of the ringforms a bond with the R¹ group (via B), or alternatively C¹ carbon atomof the ring forms a bond with the ring carbon of Formula I (via A) andthe amino substituent on the C² carbon atom of the ring forms a bondwith the R¹ group (via B). According to these embodiments, L may be anamino substituted-1,2-cyclobutane, amino substituted-1,2-oxetane, aminosubstituted-1,2-thiooxetane, or amino substituted-1,2-azetidine linkerhaving a structure:

where each R^(x) may independently be O, S, CR²CR³, or NR⁵, each R⁵ areindependently as defined herein, and the bonds A and B may be on thesame face of the ring (i.e., cis) or on opposite faces of the ring(i.e., trans).

In still other embodiments, the linker L may comprise a 1,1-cycloalkylor 1,1-heterocycle, where a carbon atom of the ring forms a bond withthe ring carbon of Formula I (via A) and the same carbon atom of thering forms a bond with the R¹ group (via B). According to certainembodiments, the 1,1-cycloalkyl may be a 3-, 4-, 5-, 6-, or 7-memberedcycloalkyl ring which may be saturated or contain one or more doublebonds. In other embodiments, the 1,1-heterocycloalkyl may be a 3-, 4-,5-, 6-, or 7-membered heterocycloalkyl ring which may be saturated orcontain one or more double bond. According to these embodiments, L maybe a 1,1-cycloalkyl or 1,1-heterocycloalkyl linker having a structure:

where m and n may each independently be an integer from 0 to 5 such thatm+n≧1 each Rx may independently be O, S, CR²CR³, or NR⁵. In thoseembodiments where the ring carbon with bonds A and B may be astereoisomer, both stereoisomers may be considered part of thedisclosure.

In still other embodiments, the linker L may comprise an aminosubstituted-1,1-cycloalkyl or an amino substituted 1,1-heterocycl, wherethe amino substituent on the C¹ carbon atom of the ring forms a bondwith the ring carbon of Formula I (via A) and C¹ carbon atom of the ringforms a bond with the R¹ group (via B), or alternatively C¹ carbon atomof the ring forms a bond with the ring carbon of Formula I (via A) andthe amino substituent on the C¹ carbon atom of the ring forms a bondwith the R¹ group (via B). According to these embodiments, L may be anamino substituted 1,1-spirocycle or an aminosubstituted1,1-spiroheterocycle linker having a structure:

where m and n may each independently be an integer from 0 to 5 such thatm+n≧1 each R^(x) may independently be O, S, CR²CR³, or NR⁵. In thoseembodiments where the ring carbon with bonds A and B may be astereoisomer, both stereoisomers may be considered part of thedisclosure.

The listing of possible structures for group L is not exhaustive and onehaving ordinary skill in the art, reading the present disclosure wouldunderstand that other possible linkers, including other possible amideisostere linkers, would also be within the scope of the structuresdescribed herein.

With respect to Formula 1 or I, Y¹ may be CR⁴ or N. In some embodiments,Y¹ is CR⁴.

With respect to Formula 1 or I, Y² may be CR⁴ or N. In some embodiments,Y² is CR⁴.

In some embodiments of the compounds represented by Formula 1 or I, Y¹and Y² are both CR⁴, and together form an additional heterocyclic ringoptionally substituted by R⁴ or R¹⁸. In some embodiments, Y¹ and Y² maytogether form a heterocyclic ring, such as an aromatic or aheteroaromatic ring, including but not limited to a thiazole ring havinga structure:

With respect to Formula 1 or I, Y³ may be CR⁴ or N. In some embodiments,Y³ is CR⁴.

With respect to Formula 1 or i, Y⁴ may be CR⁴ or N. In some embodiments,Y⁴ is CR⁴.

In some embodiments, Y¹, Y², Y³ and Y⁴ are CR⁴. In some embodiments, Y¹,Y², Y³ and Y⁴ are CH. In some embodiments, Y¹ and Y² are

and Y³ and Y⁴ are CH.

In certain embodiments of the compounds of the present disclosure, W maybe S. In certain embodiments of the present disclosure, X may be N. Inspecific embodiments, W may be S and X may be N, such that the ringcontaining W and X may be a thiazol ring. In various embodiments of thecompounds described herein, each R⁵ may independently be H or C₁₋₃alkyl.

In various embodiments of the compounds described herein, Y³ is CR⁴,wherein R⁴ is R^(b), OR^(b), COR^(b), CO₂R^(b), OCOR^(b), NR^(b)R^(c),CF₃, CN, NO₂, F, Cl, Br, or I, wherein R^(b) and R^(c) are independentlyH or C₁₋₃ alkyl. In various embodiments of the compounds describedherein, Y⁴ is CR⁴, wherein R⁴ is R^(b), OR^(b), COR^(b), CO₂R^(b),OCOR^(b), NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, or I, wherein R^(b) andR^(c) are independently H or C₁₋₃ alkyl.

In specific embodiments, R¹ may be an optionally substituted naphthylring. In certain embodiments of the present disclosure, the antiviralcompound may be a compound having a structure represented by theformula:

where A and B may independently represent a single covalent bond ordouble covalent bond, L may be a linker group comprising a structure asdescribed herein, R¹⁰, R¹³, R¹⁴, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, and R²²are independently R^(b), OR^(b), SR^(b), COR^(b), CO₂R^(b), OCOR^(b),NR^(b)R^(c), CON^(b)R^(c), NR^(b)COR^(c), SO₂NR^(b)R^(c), CF₃, CN, NO₂,F, Cl, Br, I, C₂₋₅ cyclyl or C₂₋₅ heterocyclyl and C₁₋₆ aryl or C₁₋₆heteroaryl, including where two adjacent R groups come together to forma fused cyclyl, heterocyclyl, aryl or heteroaryl ring structure; eachR^(b) is independently H or C₁₋₃ hydrocarbyl, and each R^(c) isindependently H or C₁₃ alkyl. In specific embodiments, R¹⁸ may be H orC₁₋₃ alkyl and in particular embodiments, R¹⁸ may be H.

In certain embodiments, R¹ may be a naphthyl where R¹⁹, R²⁰, R²¹, andR²² are each H, Y¹ and Y² may form a thiazole ring as shown herein whereR¹⁸ may be H, and Y³ and Y⁴ may each be CH. According to theseembodiments, the antiviral compound may be represented by the structure:

where A and B may independently represent a single covalent bond ordouble covalent bond, L may be a linker group comprising a structure asdescribed herein.

In specific embodiments, R¹ may be an optionally substituted phenylring. In certain embodiments of the present disclosure, the antiviralcompound may be a compound having a structure represented by theformula:

where A and B independently represent a single covalent bond or doublecovalent bond, L is a linker group comprising a structure as describedherein, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁶, R¹⁷, and R¹⁸ are independentlyR^(b), OR^(b), COR^(b), CO₂R^(b), OCOR^(b), NR^(b)R^(c), CF₃, CN, NO₂,F, Cl, Br, or I, wherein R^(b) and R^(c) are independently H or C₁₋₃alkyl; and, R⁵ is H or C₁₋₃ alkyl. In specific embodiments, R¹⁸ may be Hor C₁₋₃ alkyl and in particular embodiments, R¹⁸ may be CH₃. In specificembodiments, R¹³ may be halogen, and in particular embodiments, R¹³ maybe Br.

In certain embodiments, R¹ may be a phenyl where R¹⁰, R¹¹, R¹², and R¹⁴are each H, R¹³ is Br, Y¹ and Y² may form a thiazole ring as shownherein where R¹⁸ may be CH₃, and Y³ and Y⁴ may each be CH. According tothese embodiments, the antiviral compound may be represented by thestructure:

where A and B may independently represent a single covalent bond ordouble covalent bond, L may be a linker group comprising a structure asdescribed herein.

Some embodiments include compounds represented by any of Formulas 2-9.

With respect to any relevant structural feature herein, each R^(a) mayindependently be H; optionally substituted hydrocarbyl, such as C₁₋₁₂ orC₁₋₆ hydrocarbyl; optionally substituted aryl, such as optionallysubstituted C₆₋₁₂ aryl, including optionally substituted phenyl;optionally substituted heteroaryl, including optionally substitutedC₂₋₁₂ heteroaryl, such as optionally substituted pyridinyl, optionallysubstituted furyl, optionally substituted thienyl, etc. In someembodiments, each R^(a) can independently be H, or C₁₋₁₂ alkyl,including: linear or branched alkyl having the formula C_(a)H_(a+1), orcycloalkyl having the formula C_(a)H_(a−1), wherein a is 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, or 12, such as linear or branched alkyl of theformula: CH₃, C₂H₅, C₃H₇, C₄H₉, C₅H₁₁, C₆H₁₃, C₇H₁₅, C₈H₁₇, C₉H₁₉,C₁₀H₂₁, etc., or cycloalkyl of the formula: C₃H₅, C₄H₇, C₅H₉, C₆H₁₁,C₇H₁₃, C₈H₁₅, C₉H₁₇, C₁₀H₁₉, etc.

With respect to R^(a), in some embodiments, the aryl group issubstituted with halogen, trihalomethyl, alkoxy, alkylamino, OH, ON,alkylthio, arylthio, sulfoxide, arylsulfonyl, alkylsulfonyl, carboxylicacid, nitro or acylamino.

With respect to R^(a), in some embodiments, the heteroaryl group issingle or fused. In some embodiments, the single heteroaryl group isimidazole. In some embodiments, the fused heteroaryl group isbenzimidazole. In some embodiments, the heteroaryl group is substitutedwith halogen, trihalomethyl, alkoxy, alkylamino, OH, CN, alkylthio,arylthio, sulfoxide, arylsulfonyl, alkylsulfonyl, carboxylic acid, nitroor acylamino. In some embodiments, the alkyl group is branched, cyclicor polycyclic.

With respect to R^(a), a hydrocarbyl may be alkyl, alkenyl, or alkynyl.In some embodiments, the alkyl group is substituted with halogen,trihalomethyl, alkoxy, alkylamino, OH, CN, heteroaryl, alkylthio,arylthio, sulfoxide, arylsulfonyl, alkylsulfonyl, carboxylic acid,nitro, or acylamino. In some embodiments, the heteroaryl group is singleor fused. In some embodiments, the single heteroaryl group is imidazole.In some embodiments, the fused heteroaryl group is benzimidazole. Insome embodiments, the alkenyl group is branched, cyclic or polycyclic.In some embodiments, the alkenyl group is substituted with halogen,trihalomethyl, alkoxy, alkylamino, OH, CN, heteroaryl, alkylthio,arylthio, sulfoxide, arylsulfonyl, alkylsulfonyl, carboxylic acid,nitro, or acylamino.

With respect to any relevant structural feature herein, R^(b) may be H,or C₁₋₃ hydrocarbyl, such as CH₃, C₂H₅, C₃H₇, cyclopropyl, CH═CH₂,CH₂CH═CH₂, C≡CH, CH₂C≡CH, etc.

With respect to any relevant structural feature herein, R^(c) may be H,or C₁₋₃ alkyl, such as CH₃, C₂H₅, C₃H₇, cyclopropyl, etc. In someembodiments, R^(c) is H.

With respect to any relevant formula or structural depiction herein,such as Formula I, Formula II, Formula III, Formula IV, or Formula V, R¹may be R^(a), OR² or NR²R³. In some embodiments, R¹ may be optionallysubstituted phenyl. In some embodiments, R¹ may be unsubstituted phenyl.In some embodiments, R¹ may be substituted or unsubstituted pyridyl orpyrimidyl. In some embodiments, R¹ may be optionally substitutednaphthyl. In some embodiments, R¹ may be unsubstituted naphthyl. In someembodiments, R¹ may be substituted or unsubstituted quinolinyl,isoquinolinyl, or azoquinolinyl.

In some embodiments, R¹ may be

In some embodiments, R¹ may be

In some embodiments, R¹ may be

In some embodiments, R¹ may be

In some embodiments, R¹ may be

In some embodiments, R¹ may be

In some embodiments, R¹ may be

With respect to any relevant structural feature herein, R² may be R^(a),COR^(a), or SO₂R^(a). In some embodiments, R² may be H, methyl, ethyl, apropyl (e.g. n-propyl, isopropyl, etc.), cyclopropyl, a butyl,cyclobutyl or an isomer thereof, a pentyl, cyclopentyl or an isomerthereof, a hexyl, a cyclohexyl or an isomer thereof, etc. In someembodiments, R² may be H.

With respect to any relevant structural feature herein, R³ may be R^(a),COR^(a), or SO₂R^(a). In some embodiments, R³ may be H, methyl, ethyl, apropyl (e.g. n-propyl, isopropyl, etc.), cyclopropyl, a butyl,cyclobutyl or an isomer thereof, a pentyl, cyclopentyl or an isomerthereof, a hexyl, a cyclohexyl or an isomer thereof, etc. In someembodiments, R³ may be H.

With respect to any relevant structural feature herein, each R⁴ mayindependently be R², OR^(a), COR^(a), CO₂R^(a), OCOR^(a), CONR²R³,NR²R³, NR^(b)COR^(a), SR^(a), SOR^(a), SO₂R^(a), SO₂NR^(a)R^(b),NCOR^(a), halogen, trihalomethyl, CN, S═O, nitro, or C₂₋₅ heteroaryl. Insome embodiments, R⁴ may be H.

Generally R⁵ and R⁸-R³², may be H or any substituent, such as asubstituent having from 0 to 6 carbon atoms and from 0 to 5 heteroatomsindependently selected from: O, N, S, F, Cl, Br, and I, and/or having amolecular weight of 15 g/mol to 300 g/mol. Any of R⁵ and R⁸-R³² maycomprise: a) 1 or more alkyl moieties optionally substituted with, oroptionally connected by, b) 1 or more functional groups, such as C═C,C≡C, CO, CO₂, CON, NCO₂, OH, SH, O, S, N, N═C, F, Cl, Br, I, CN, NO₂,CO₂H, NH₂, etc.; or may be a substituent having no alkyl portion, suchas F, Cl, Br, I, NO₂, CN, NH₂, OH, COH, CO₂H, etc.

With respect to any relevant structural feature herein, In someembodiments, R⁵ may be R^(a), COR^(a), SO₂R^(a), or may not be present.Some non-limiting examples of R⁵ may include H or C₁₋₃ alkyl, such asCH₃, C₂H₅, C₇, cyclopropyl, etc. In some embodiments, R⁵ may be CH₃ Insome embodiments, R⁵ may be H.

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R⁸ may include R^(b), OR^(b), SR^(b), COR^(b),CO₂R^(b), OCOR^(b), NR^(b)R^(c), CON^(b)R^(c), NR^(b)COR^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R⁸ may be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂,NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C≡CH, or NO₂. In someembodiments, R⁸ may be H.

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R⁹ may include R^(b), COR^(b), CO₂R^(b),CON^(b)R^(c), NR^(b)COR^(c), SO₂NR^(b)R^(c), CF₃, CN, or C₂₋₅heterocyclyl. In some embodiments, R⁹ may be H, CH₃, CH₂CH₃, SO₂NH₂, orCH₂C≡CH. In some embodiments, R⁹ may be H, CH₃, CH₂CH₃, CH₂CH₂CH₃,CH₂CH═CH₂, or CH₂C≡CH. In some embodiments, R⁹ may be CH₂C≡CH. In someembodiments, R⁹ may be H.

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R¹⁰ may include R^(b), OR^(b), SR^(b), COR^(b),CO₂R^(b), OCOR^(b), NR^(b)R^(c), CON^(b)R^(c), NR^(b)COR^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R¹⁰ may be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃,NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C≡CH, or NO₂. In someembodiments, R¹⁰ may be H.

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R¹¹ may include R^(b), OR^(b), SR^(b), COR^(b),CO₂R^(b), OCOR^(b), NR^(b)R^(c), CON^(b)R^(c), NR^(b)COR^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R¹¹ may be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃,NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C≡CH, or NO₂. In someembodiments, R¹¹ may be H, Cl or Br. In some embodiments, R¹¹ may be Cl.In some embodiments, R¹¹ may be Br. In some embodiments, R11 may be H.

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R¹² may include R^(b), OR^(b), SR^(b), COR^(b),CO₂R^(b), OCOR^(b), NR^(b)R^(c), CON^(b)R^(c), NR^(b)COR^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R¹² may be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃,NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C≡CH, or NO₂. In someembodiments, R¹² may be H, Cl, or SO₂NH₂. In some embodiments, R¹² maybe H. In some embodiments, R¹² may be Cl. In some embodiments, R¹² maybe SO₂NH₂. In some embodiments, R¹² may be H.

In some embodiments, R¹¹ and R¹² may together form a fused cyclic,heterocyclic, aryl, or heteroaryl structure, such as, but not limited tothe structure:

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R¹³ may include R^(b), OR^(b), SR^(b), COR^(b),CO₂R^(b), OCOR^(b), NR^(b)R^(c), CON^(b)R^(c), NR^(b)COR^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R¹³ may be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃,NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C≡CH, or NO₂. In someembodiments, R¹³ may be H or Cl. In some embodiments, R¹³ may be H. Insome embodiments, R¹³ may be Cl. In some embodiments, R¹¹ and R¹³ mayeach be Cl.

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R¹⁴ may include R^(b), OR^(b), SR^(b), COR^(b),CO₂R^(b), OCOR^(b), NR^(b)R^(c), CON^(b)R^(c), NR^(b)COR^(c), CF₃, CN,NO₂, F, Cl, Br, or I. In some embodiments, R¹⁴ may be H, CH₃, CH₂CH₃,Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂, CH₂C≡CH, or NO₂. In someembodiments, R¹⁴ may be H.

In some embodiments, R¹⁰ and R¹⁴ may be H. In some embodiments, R¹⁰,R¹², and R¹⁴ may be H. In some embodiments, R¹⁰, R¹³, and R¹⁴ may be H.In some embodiments, R¹⁰, R¹¹, R¹³, and R¹⁴ may be H. In someembodiments, R¹⁰, R¹¹, R¹², and R¹⁴ may be H. In some embodiments, R¹⁰,R¹¹, R¹², R¹³, and R¹⁴ may be H.

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R¹⁶ may include R^(b), OR^(b), SR^(b), COR^(b),CO₂R^(b), OCOR^(b), NR^(b)R^(c), CON^(b)R^(c), NR^(b)COR^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, or C₂₋₅ heterocyclyl. In someembodiments, R¹⁶ may be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂,NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C≡CH, or NO₂. In someembodiments, R¹⁶ may be H.

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R¹⁷ may include R^(b), OR^(b), SR^(b), COR^(b),CO₂R^(b), OCOR^(b), NR^(b)R^(c), CON^(b)R^(c), NR^(b)COR^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R¹⁷ may be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃,NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C≡CH, or NO₂. In someembodiments, R¹⁷ may be H.

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R¹⁸ may include R^(b), OR^(b), SR^(b), COR^(b),CO₂R^(b), OCOR^(b), NR^(b)R^(c), CON^(b)R^(c), NR^(b)COR^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R¹⁸ may be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃,NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C≡CH, or NO₂. In someembodiments, R¹⁸ may be H or CH₃. In some embodiments, R¹⁸ may be H. Insome embodiments, R¹⁸ may be CH₃.

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R¹⁹ may include R^(b), OR^(b), SR^(b), COR^(b),CO₂R^(b), OCOR^(b), NR^(b)R^(c), CON^(b)R^(c), NR^(b)COR^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, or C₂₋₅ heterocyclyl. In someembodiments, R¹⁹ may be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂,NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C≡CH, or NO₂. In someembodiments, R¹⁹ may be H.

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R²⁰ may include R^(b), OR^(b), SR^(b), COR^(b),CO₂R^(b), OCOR^(b), NR^(b)R^(c), CON^(b)R^(c), NR^(b)COR^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R²⁰ may be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃,NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, CH₂C≡CH, or NO₂. In some embodiments, R²⁰may be H.

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R²¹ may include R^(b), OR^(b), SR^(b), COR^(b),CO₂R^(b), OCOR^(b), NR^(b)R^(c), CON^(b)R^(c), NR^(b)COR^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R²¹ may be H, CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃,N(CH₃)₂, SO₂NH₂, morpholino, CH₂C≡CH, or NO₂. In some embodiments, R²¹may be H.

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R²² may include R^(b), OR^(b), SR^(b), COR^(b),CO₂R^(b), OCOR^(b), NR^(b)R^(c), CON^(b)R^(c), NR^(b)COR^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R²² may be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃,NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, or NO₂. In some embodiments,R²² may be H.

In some embodiments, R¹⁹, R²⁰, R²¹, and R²² may be H.

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R²³ may include R^(b), OR^(b), SR^(b), COR^(b),CO₂R^(b), OCOR^(b), NR^(b)R^(c), CON^(b)R^(c), NR^(b)COR^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R²³ may be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃,NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, CH₂C≡CH, or NO₂. In some embodiments, R²³may be H or SO₂NH₂. In some embodiments, R²³ may be H. In someembodiments, R²³ may be SO₂NH₂.

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R²⁴ may include R^(b), OR^(b), SR^(b), COR^(b),CO₂R^(b), OCOR^(b), NR^(b)R^(c), CON^(b)R^(c), NR^(b)COR^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R²⁴ may be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃,NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, or NO₂. In some embodiments,R²⁴ may be H.

In some embodiments, R²³ and R²⁴ may be H.

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R²⁵ may include R^(b), OR^(b), SR^(b), COR^(b),CO₂R^(b), OCOR^(b), NR^(b)R^(c), CON^(b)R^(c), NR^(b)COR^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, or C₂₋₅ heterocyclyl. In someembodiments, R²⁵ may be H, CH₃, CH₂CH₃, N(CH₃)₂, SO₂NH₂, morpholino,CH₂C≡CH, or NO₂. In some embodiments, R²⁵ may be CH₃ or H. In someembodiments, R²⁵ may be H.

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R²⁶ may include R^(b), CF₃, CN, or NO₂. In someembodiments, R²⁶ is H, CH₃, or CH₂CH₃. In some embodiments, R²⁶ may beH.

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R²⁷ may include R^(b), OR^(b), SR^(b), COR^(b),CO₂R^(b), CON^(b)R^(c), SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, orC₂₋₅ heterocyclyl. In some embodiments, R²⁷ may be H, CH₃, CH₂CH₃, Cl,Br, OH, OCH₃, SCH₃, SO₂NH₂, CH₂C≡CH, or NO₂. In some embodiments, R²⁷may be H, (CH₂)₃CH₃, CH₂CH₂OCH₃, CH₂CH₂N(CH₃)₂, CH₂CH₂-morpholino, orCH₂CH₂SCH₃. In some embodiments, R²⁷ may be H. In some embodiments, R²⁷may be (CH₂)₃CH₃. In some embodiments, R²⁷ may be CH₂CH₂OCH₃. In someembodiments, R²⁷ may be CH₂CH₂N(CH₃)₂. In some embodiments, R²⁷ may beCH₂CH₂-morpholino. In some embodiments, R²⁷ may be CH₂CH₂SCH₃.

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R²⁸ may include R^(b), OR^(b), SR^(b), COR^(b),CO₂R^(b), OCOR^(b), NR^(b)R^(c), CON^(b)R^(c), NR^(b)COR^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R²⁸ may be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃,NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C≡CH, or NO₂. In someembodiments, R²⁸ may be H, CH₂CH₃, OCH₃, N(CH₃)₂, morpholino, or SCH₃.In some embodiments, R²⁸ may be H. In some embodiments, R²⁸ may beCH₂CH₃. In some embodiments, R²⁸ may be OCH₃. In some embodiments, R²⁸may be CN(CH₃)₂. In some embodiments, R²⁸ may be morpholino. In someembodiments, R²⁸ may be SCH₃.

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R²⁹ may include R^(b), OR^(b), SR^(b), CF₃, CN,NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. In some embodiments, R²⁹ may beH, CH₃, or CH₂CH₃. In some embodiments, R²⁹ may be H.

In some embodiments, R⁸ and R²⁹ may be H.

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R³⁰ may include R^(b), OR^(b), SR^(b), COR^(b),CO₂R^(b), OCOR^(b), NR^(b)R^(c), CON^(b)R^(c), NR^(b)COR^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, or I. In some embodiments, R³⁰may be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃, NH₂, NHCH₃, N(CH₃)₂,SO₂NH₂, CH₂C≡CH, or NO₂. In some embodiments, R³⁰ may be H.

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R³¹ may include R^(b), OR^(b), SR^(b), COR^(b),CO₂R^(b), OCOR^(b), NR^(b)R^(c), CON^(b)R^(c), NR^(b)COR^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R³¹ may be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃,NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C≡CH, or NO₂. In someembodiments, R³¹ may be H.

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R³² may include R^(b), OR^(b), SR^(b), COR^(b),CO₂R^(b), OCOR^(b), NR^(b)R^(c), CON^(b)R^(c), NR^(b)COR^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R³² may be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃,NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C≡CH, or NO₂. In someembodiments, R³² may be H or NO₂. In some embodiments, R³² may be H. Insome embodiments, R³² may be NO₂.

In some embodiments, R³⁰, R³¹, and R³² may be H. In some embodiments,R³¹ and R³² may be H.

With respect to any relevant formula or structural depiction above, somenon-limiting examples of R³³ may include R^(b), OR^(b), SR^(b), COR^(b),CO₂R^(b), OCOR^(b), NR^(b)R^(c), CON^(b)R^(c), NR^(b)COR^(c),SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅ heterocyclyl. Insome embodiments, R³² may be H, CH₃, CH₂CH₃, Cl, Br, OH, OCH₃, SCH₃,NH₂, NHCH₃, N(CH₃)₂, SO₂NH₂, morpholino, CH₂C≡CH, or NO₂. In someembodiments, R³³ may be H.

Unless otherwise indicated, any reference to a compound herein bystructure, formula, name or any other means, includes pharmaceuticallyacceptable salts, such as sodium, potassium, and ammonium salts;prodrugs, such as ester prodrugs; alternate solid forms, such aspolymorphs, solvates, hydrates, etc.; tautomers; or, any other chemicalspecies that may rapidly convert to a compound described herein underconditions in which the compounds are used as described herein.

Unless stereochemistry is unambiguously depicted, any structure, formulaor name for a compound can refer to any stereoisomer or any mixture ofstereoisomers of the compound.

As used herein, the term “functional group” refers to an atom or a groupof atoms that have similar chemical properties whenever they occur indifferent compounds, and as such the functional group defines thecharacteristic physical and chemical properties of families of organiccompounds.

Unless otherwise indicated, when any compound or chemical structuralfeature (collectively referred to herein as a “compound”), such as forexample alkyl, aryl, etc., is referred to as being “optionallysubstituted,” that compound can have no substituents (in which case itis “unsubstituted”), or it can include one or more substituents (inwhich case it is “substituted”). The term “substituent” has the ordinarymeaning known to one of ordinary skill in the art. In some embodiments,the substituent may be an ordinary organic moiety known in the art,which can have a molecular weight (e.g., the sum of the atomic masses ofthe atoms of the substituent) of 15 g/mol to 50 g/mol, 15 g/mol to 100g/mol, 15 g/mol to 150 g/mol, 15 g/mol to 200 g/mol, 15 g/mol to 300g/mol, or 15 g/mol to 500 g/mol. In some embodiments, the substituentcomprises: 0-30, 0-20, 0-10, or 0-5 carbon (C) atoms; and/or 0-30, 0-20,0-10, or 0-5 heteroatoms including N, O, S, Si, F, Cl, Br, or I;provided that the substituent comprises at least one atom including C,N, O, S, Si, F, Cl, Br, or I in a substituted compound. Examples ofsubstituents include, but are not limited to, alkyl, alkenyl, alkynyl,heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, hydroxy,alkoxy, aryloxy, acyl, acyloxy, alkylcarboxylate, thiol, alkylthio,cyano, halo, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido,isocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl,sulfinyl, sulfonyl, haloalkyl, haloalkoxyl, trihalomethanesulfonyl,trihalomethanesulfonamido, amino, etc. For convenience, the term“molecular weight” is used with respect to a moiety or part of amolecule to indicate the sum of the atomic masses of the atoms in themoiety or part of a molecule, even though it may not be a completemolecule.

As used herein, the term “hydrocarbyl” has the broadest meaninggenerally understood in the art, and can include a moiety composed ofcarbon and hydrogen. Some examples can include alkyl, alkenyl, alkynyl,aryl, etc., and combinations thereof, and can be linear, branched,cyclic, or a combination thereof. Hydrocarbyl can be bonded to any othernumber of moieties (for example, can be bonded to one other group, suchas —CH₃, —CH═CH₂, etc.; two other groups, such as -phenyl-, —C≡C—, etc.;or any number of other groups) that the structure can bear, and in someembodiments, can contain from one to thirty-five carbon atoms. Examplesof hydrocarbyl groups include but are not limited to C₁ alkyl, C₂ alkyl,C₂ alkenyl, C₂ alkynyl, C₃ alkyl, C₃ alkenyl, C₃ alkynyl, C₄ alkyl, C₄alkenyl, C₄ alkynyl, C₅ alkyl, C₅ alkenyl, C₅ alkynyl, C₆ alkyl, C₆alkenyl, C₆ alkynyl, phenyl, etc.

As used herein the term “alkyl” has the broadest meaning generallyunderstood in the art, and can include a moiety composed of carbon andhydrogen containing no double or triple bonds and not having any cyclicstructure. Alkyl can be linear alkyl, branched alkyl, cycloalkyl, or acombination thereof, and in some embodiments, can contain from one tothirty-five carbon atoms. In some embodiments, alkyl can include C₁₋₁₀linear alkyl, such as methyl (—CH₃), ethyl (—CH₂CH₃), n-propyl(—CH₂CH₂CH₃), n-butyl (—CH₂CH₂CH₂CH₃), n-pentyl (—CH₂CH₂CH₂CH₂CH₃),n-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), etc.; C₃₋₁₀ branched alkyl, such as C₃H₇(e.g. iso-propyl), C₄H₉ (e.g., branched butyl isomers), C₅H₁₁ (e.g.,branched pentyl isomers), C₆H₁₃ (e.g., branched hexyl isomers), C₇H₁₅(e.g., branched heptyl isomers), etc.; C₃₋₁₀ cycloalkyl, such as C₃H₅(e.g. cyclopropyl), C₄H₇ (e.g., cyclobutyl isomers such as cyclobutyl,methylcyclopropyl, etc.), C₅H₉ (e.g., cyclopentyl isomers such ascyclopentyl, methylcyclobutyl, dimethylcyclopropyl, etc.) C₆H₁₁ (e.g.,cyclohexyl isomers), C₇H₁₃ (e.g., cycloheptyl isomers), etc.; and thelike.

The terms “alkyl,” “alkenyl” and “alkynyl” refer to substituted andunsubstituted alkyls, alkenyls and alkynyls, respectively. An alkylgroup can be optionally substituted as defined herein.

Substituted alkyls, alkenyls and alkynyls refers to alkyls, alkenyls andalkynyls substituted with one to five substituents including H, loweralkyl, aryl, alkenyl, alkynyl, arylalkyl, alkoxy, aryloxy, arylalkoxy,alkoxyalkylaryl, alkylamino, arylamino, NH₂, OH, CN, NO₂, OCF₃, CF₃, F,1-amidine, 2-amidine, alkylcarbonyl, morpholinyl, piperidinyl, dioxanyl,pyranyl, heteroaryl, furanyl, thiophenyl, tetrazolo, thiazolyl,isothiazolyl, imidazolyl, thiadiazolyl, thiadiazole S-oxide, thiadiazoleS,S-dioxide, pyrazolo, oxazolyl, isoxazolyl, pyridinyl, pyrimidinyl,quinolinyl, isoquinolinyl, SR, SOR, SO₂R, CO₂R, COR, CONR′R″, CSNR′R″and SO_(n)NR′R″.

As used herein, either alone or in combination, the term “alkynyl”refers to a functional group comprising a straight-chain orbranched-chain hydrocarbon containing from 2 to 20 carbon atoms andhaving one or more carbon-carbon triple bonds and not having any cyclicstructure. An alkynyl group may be optionally substituted as definedherein. Examples of alkynyl groups include, without limitation, ethynyl,propynyl, hydroxypropynyl, butynyl, butyn-1-yl, butyn-2-yl,3-methylbutyn-1-yl, pentynyl, pentyn-1-yl, hexynyl, hexyn-2-yl,heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl, tridecynyl,tetradecynyl, pentadecynyl, hexadecynyl, heptadecynyl, octadecynyl,nonadecynyl, eicosynyl, and the like.

The term “alkylene” as used herein, alone or in combination, refers to asaturated aliphatic group derived from a straight or branched chainsaturated hydrocarbon attached at two or more positions, such asmethylene (—CH₂—). Unless otherwise specified, the term “alkyl” mayinclude “alkylene” groups.

As used herein, either alone or in combination, the term “alkylcarbonyl”or “alkanoyl” refers to a functional group comprising an alkyl groupattached to the parent molecular moiety through a carbonyl group.Examples of alkylcarbonyl groups include, without limitation,methylcarbonyl, ethylcarbonyl, and the like.

As used herein, either alone or in combination, the term “heteroalkyl”refers to a functional group comprising a straight-chain orbranched-chain hydrocarbon containing from 1 to 20 atoms linkedexclusively by single bonds, where at least one atom in the chain is acarbon and at least one atom in the chain is O, S, N, or any combinationthereof. The heteroalkyl group can be fully saturated or contain from 1to 3 degrees of unsaturation. The non-carbon atoms can be at anyinterior position of the heteroalkyl group, and up to two non-carbonatoms may be consecutive, such as, e.g., —CH₂—NH—OCH₃. In addition, thenon-carbon atoms may optionally be oxidized and the nitrogen mayoptionally be quaternized.

As used herein, either alone or in combination, the term “alkyloxy” or“alkoxy” refers to a functional group comprising an alkyl ether group.Examples of alkoxys include, without limitation, methoxy, ethoxy,n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy,and the like.

As used herein, either alone or in combination, the term “hydroxy”refers to the functional group hydroxyl (—OH).

As used herein, either alone or in combination, the term “carboxyl” or“carboxy” refers to the functional group —C(═O)OH or the corresponding“carboxylate” anion —C(═O)O—. Examples include, without limitation,formic acid, acetic acid, oxalic acid, benzoic acid. An “O-carboxyl”group refers to a carboxyl group having the general formula RCOO,wherein R is an organic moiety or group. A “C-carboxyl” group refers toa carboxyl group having the general formula COOR, wherein R is anorganic moiety or group.

As used herein, either alone or in combination, the term “oxo” refers tothe functional group ═O.

As used herein, the term “carbocyclic” has the broadest meaninggenerally understood in the art, and includes a ring or ring systemwherein the ring atoms are all carbon. Examples include, but are notlimited to, phenyl, naphthyl, anthracenyl, cycloalkyl, cycloalkenyl,cycloalkynyl, etc., and combinations thereof.

As used herein, the term “heterocyclic” has the broadest meaninggenerally understood in the art, and includes a ring or ring systemwherein at least one of the ring atoms is not carbon, such as N, O, S,etc. Examples include, but are not limited to, heteroaryl,cycloheteroalkyl, cycloheteroalkenyl, cycloheteroalkynyl, etc., andcombinations thereof.

As used herein, either alone or in combination, the term “cycloalkyl,”“carbocyclicalkyl” and “carbocycloalkyl” refers to a functional groupcomprising a substituted or unsubstituted non-aromatic hydrocarbon witha non-conjugated cyclic molecular ring structure of 3 to 12 carbon atomslinked exclusively with carbon-carbon single bonds in the carbon ringstructure. A cycloalkyl group can be monocyclic, bicyclic or polycyclic,and may optionally include one to three additional ring structures, suchas, e.g., an aryl, a heteroaryl, a cycloalkenyl, a heterocycloalkyl, ora heterocycloalkenyl.

As used herein, either alone or in combination, the term “lowercycloalkyl” refers to a functional group comprising a monocyclicsubstituted or unsubstituted non-aromatic hydrocarbon with anon-conjugated cyclic molecular ring structure of 3 to 6 carbon atomslinked exclusively with carbon-carbon single bonds in the carbon ringstructure. Examples of lower cycloalkyl groups include, withoutlimitation, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

As used herein the term “aryl” has the broadest meaning generallyunderstood in the art, and can include an aromatic ring or aromatic ringsystem. An aryl group can be monocyclic, bicyclic or polycyclic, and mayoptionally include one to three additional ring structures; such as, forexample, a cycloalkyl, a cycloalkenyl, a heterocycloalkyl, aheterocycloalkenyl, or a heteroaryl. The term “aryl” includes, withoutlimitation, phenyl(benzenyl), thiophenyl, indolyl, naphthyl, tolyl,xylyl, anthracenyl, phenanthryl, azulenyl, biphenyl, naphthalenyl,1-methylnaphthalenyl, acenaphthenyl, acenaphthylenyl, anthracenyl,fluorenyl, phenalenyl, phenanthrenyl, benzo[a]anthracenyl,benzo[c]phenanthrenyl, chrysenyl, fluoranthenyl, pyrenyl, tetracenyl(naphthacenyl), triphenylenyl, anthanthrenyl, benzopyrenyl,benzo[a]pyrenyl, benzo[e]fluoranthenyl, benzo[ghi]perylenyl,benzo[j]fluoranthenyl, benzo[k]fluoranthenyl, corannulenyl, coronenyl,dicoronylenyl, helicenyl, heptacenyl, hexacenyl, ovalenyl, pentacenyl,picenyl, perylenyl, tetraphenylenyl, etc.

Additionally, as used herein, either alone or in combination, the term“aryl,” “hydrocarbyl aryl” or “aryl hydrocarbon” can refer to afunctional group comprising a substituted or unsubstituted aromatichydrocarbon with a conjugated cyclic molecular ring structure of 3 to 12carbon atoms. Substituted aryl refers to aryls substituted with one tofive substituents including H, lower alkyl, aryl, alkenyl, alkynyl,arylalkyl, alkoxy, aryloxy, arylalkoxy, alkoxyalkylaryl, alkylamino,arylamino, NH₂, OH, CN, NO₂, OCF₃, CF₃, Br, Cl, F, 1-amidino, 2-amidino,alkylcarbonyl, morpholino, piperidinyl, dioxanyl, pyranyl, heteroaryl,furanyl, thiophenyl, tetrazolo, thiazole, isothiazolo, imidazolo,thiadiazole, thiadiazole S-oxide, thiadiazole S,S-dioxide, pyrazolo,oxazole, isoxazole, pyridinyl, pyrimidinyl, quinoline, isoquinoline, SR,SOR, SO₂R, CO₂R, COR, CONR′R″, CSNR′R″, SO_(n)NR′R″, etc.

As used herein, either alone or in combination, the term “lower aryl”refers to a functional group comprising a substituted or unsubstitutedaromatic hydrocarbon with a conjugated cyclic molecular ring structureof 3 to 6 carbon atoms. Examples of lower aryl groups include, withoutlimitation, phenyl and naphthyl.

As used herein, either alone or in combination, the term “heteroaryl”refers to a functional group comprising a substituted or unsubstitutedaromatic hydrocarbon with a conjugated cyclic molecular ring structureof 3 to 12 atoms, where at least one atom in the ring structure is acarbon and at least one atom in the ring structure is O, S, N, or anycombination thereof. A heteroaryl group can be monocyclic, bicyclic orpolycyclic, and may optionally include one to three additional ringstructures, such as, e.g., an aryl, a cycloalkyl, a cycloalkenyl, aheterocycloalkyl, or a heterocycloalkenyl. Examples of heteroaryl groupsinclude, without limitation, acridinyl, benzidolyl, benzimidazolyl,benzisoxazolyl, benzodioxinyl, dihydrobenzodioxinyl, benzodioxolyl,1,3-benzodioxolyl, benzofuryl, benzoisoxazolyl, benzopyranyl,benzothiophenyl, benzo[c]thiophenyl, benzotriazolyl, benzoxadiazolyl,benzoxazolyl, benzothiadiazolyl, benzothiazolyl, benzothienyl,carbazolyl, chromonyl, cinnolinyl, dihydrocinnolinyl, coumarinyl,dibenzofuranyl, furopyridinyl, furyl, indolizinyl, indolyl,dihydroindolyl, imidazolyl, indazolyl, isobenzofuryl, isoindolyl,isoindolinyl, dihydroisoindolyl, isoquinolyl, dihydroisoquinolinyl,isoxazolyl, isothiazolyl, oxazolyl, oxadiazolyl, phenanthrolinyl,phenanthridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridyl,pyrimidinyl, pyridazinyl, pyrrolinyl, pyrrolyl, pyrrolopyridinyl,quinolyl, quinoxalinyl, quinazolinyl, tetrahydroquinolinyl,tetrazolopyridazinyl, tetrahydroisoquinolinyl, thiophenyl, thiazolyl,thiadiazolyl, thienopyridinyl, thienyl, thiophenyl, triazolyl,xanthenyl, and the like.

As used herein, either alone or in combination, the term “lowerheteroaryl” refers to a functional group comprising a monocyclic orbicyclic, substituted or unsubstituted aromatic hydrocarbon with aconjugated cyclic molecular ring structure of 3 to 6 atoms, where atleast one atom in the ring structure is a carbon and at least one atomin the ring structure is O, S, N, or any combination thereof.

The phenyl structure associated with some of the embodiments describedherein is depicted below. This structure can be unsubstituted, as shownbelow, or can be substituted such that a substituent can independentlybe in any position normally occupied by a hydrogen atom when thestructure is unsubstituted. Unless a point of attachment is indicated bybond to a specific carbon atom, attachment may occur at any positionnormally occupied by a hydrogen atom.

Each R^(a) can independently be H; optionally substituted hydrocarbyl;optionally substituted aryl, such as optionally substituted phenyl oroptionally substituted aryl; optionally substituted heteroaryl, such asoptionally substituted pyridinyl, optionally substituted furyl,optionally substituted thienyl, etc. In some embodiments, each R^(a) canindependently be H, or C₁₋₁₂ alkyl, including: linear or branched alkylhaving the formula C_(a)H_(a+1), or cycloalkyl having the formulaC_(a)H_(a−1), wherein a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12,such as linear or branched alkyl of the formula: CH₃, C₂H₅, C₃H₇, C₄H₉,C₅H₁₁, C₆H₁₃, C₇H₁₅, C₈H₁₇, C₉H₁₉, C₁₀H₂₁, etc., or cycloalkyl of theformula: C₃H₅, C₄H₇, C₅H₉, C₆H₁₁, C₇H₁₃, C₈H₁₅, C₉H₁₇, C₁₀H₁₉, etc.

The term “treat” includes one or more of the diagnosis, cure,mitigation, vaccination, augmentation of a therapy or prevention ofdisease in man or other animals

As used herein, the term “vertebrate” includes all living vertebratessuch as, without limitation, mammals, humans, birds, dogs, cats,livestock, farm animals, free-range herds, etc.

Many RNA viruses share biochemical, regulatory, and signaling pathways.These viruses include but are not limited to influenza virus (includingavian and swine isolates), respiratory syncytial virus, Hepatitis Cvirus, West Nile virus, SARS-coronavirus, poliovirus, measles virus,Dengue virus, yellow fever virus, tick-borne encephalitis virus,Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valleyvirus, Powassan virus, Rocio virus, louping-ill virus, Banzi virus,Ilheus virus, Kokobera virus, Kunjin virus, Alfuy virus, bovine diarrheavirus, and the Kyasanur forest disease virus. The compounds and methodsdisclosed herein can be used to treat these viruses.

Relevant taxonomic families of RNA viruses include, without limitation,Arenaviridae, Astroviridae, Birnaviridae, Bromoviridae, Bunyaviridae,Caliciviridae, Closteroviridae, Comoviridae, Cystoviridae, Flaviviridae,Flexiviridae, Hepevirus, Leviviridae, Luteoviridae, Mononegavirales,Mosaic Viruses, Nidovirales, Nodaviridae, Orthomyxoviridae,Paramyxoviridae, Picobirnavirus, Picornaviridae, Potyviridae,Reoviridae, Retroviridae, Sequiviridae, Tenuivirus, Togaviridae,Tombusviridae, Totiviridae, and Tymoviridae. The compounds and methodsdisclosed herein can be used to treat viruses within these families ofviruses as part of a pharmaceutically acceptable drug formulation. Otherrelevant virus families include, without limitation, Hepadnaviridae,Herpesviridae, and Papillomaviridae.

Particular embodiments provide for pharmaceutical compositionscomprising the compounds, alone or in combination with an antigen, forthe purpose of treating and/or preventing disease in an animal includinga vertebrate animal. As such, in some embodiments the pharmaceuticalcompositions can be used as vaccines.

The disclosure provides for the use of the compounds as adjuvants.

The compounds and methods disclosed herein can be additive orsynergistic with other therapies currently in development or use. Forexample, ribavirin and interferon-α provide an effective treatment forHCV infection when used in combination. Their efficacy in combinationcan exceed the efficacy of either drug product when used alone. Thecompositions of the disclosure can be administered alone or incombination or conjunction with interferon, ribavirin and/or a varietyof small molecules that are being developed against both viral targets(viral proteases, viral polymerase, assembly of viral replicationcomplexes) and host targets (host proteases required for viralprocessing, host kinases required for phosphorylation of viral targetssuch as NS5A, and inhibitors of host factors required to efficientlyutilize the viral internal ribosome entry site, or IRES).

The compounds and methods disclosed herein could be used in combinationor conjunction with, without limitation, adamantane inhibitors,neuraminidase inhibitors, alpha interferons, non-nucleoside ornucleoside polymerase inhibitors, NS5A inhibitors, antihistamines,protease inhibitors, helicase inhibitors, P7 inhibitors, entryinhibitors, IRES inhibitors, immune stimulators, HCV replicationinhibitors, cyclophilin A inhibitors, A3 adenosine agonists, andmicroRNA suppressors.

Cytokines that could be administered in combination or conjunction withthe compounds and methods disclosed herein include, without limitation,IL-2, IL-12, IL-23, IL-27, or IFN-γ. New HCV drugs that are or will beavailable for potential administration in combination or conjunctionwith the compounds and methods disclosed herein include, withoutlimitation, ACH-1625 (Achillion); Glycosylated interferon (AliosBiopharma); ANA598, ANA773 (Anadys Pharm); ATI-0810 (ArisynTherapeutics); AVL-181 (Avila Therapeutics); LOCTERON® (Biolex,Pittsboro, N.C.); CTS-1027 (Conatus); SD-101 (Dynavax Technologies);Clemizole (Eiger Biopharmaceuticals); GS-9190 (Gilead Sciences); GI-5005(GlobalImmune BioPharma); Resiquimod/R-848 (Graceway Pharmaceuticals);Albinterferon alpha-2b (Human Genome Sciences); IDX-184, IDX-320,IDX-375 (Idenix); IMO-2125 (Idera Pharmaceuticals); INX-189 (Inhibitex);ITCA-638 (Intarcia Therapeutics); ITMN-191/RG7227 (Intermune); ITX-5061,ITX-4520 (iTherx Pharmaceuticals); MB11362 (Metabasis Therapeutics);Bavituximab (Peregrine Pharmaceuticals); PSI-7977, RG7128, PSI-938(Pharmasset); PHX1766 (Phenomix); Nitazoxanide/ALINIA® (RomarkLaboratories, Tampa, Fla.); SP-30 (Samaritan Pharmaceuticals); SCV-07(SciClone); SCY-635 (Scynexis); TT-033 (Tacere Therapeutics);Viramidine/taribavirin (Valeant Pharmaceuticals); Telaprevir, VCH-759,VCH-916, VCH-222, VX-500, VX-813 (Vertex Pharmaceuticals); and PEG-INFLambda (Zymogenetics).

New influenza and West Nile virus drugs that are or will be availablefor potential administration in combination or conjunction with thecompounds and methods disclosed herein include, without limitation,neuraminidase inhibitors (Peramivir, Laninamivir); tripletherapy—neuraminidase inhibitors ribavirin, amantadine (ADS-8902);polymerase inhibitors (Favipiravir); reverse transcriptase inhibitor(ANX-201); inhaled chitosan (ANX-211); entry/binding inhibitors (BindingSite Mimetic, FLUCIDE™ (NanoViricides, West Haven, Conn.); entryinhibitor (FLUDASE® (NexBio, San Diego, Calif.); fusion inhibitor,(MGAWN1 for West Nile); host cell inhibitors (lantibiotics); cleavage ofRNA genome (RNAi, RNAse L); immune stimulators (Interferon, Alferon-LDO;Neurokinin1 agonist, Homspera, Interferon Alferon N for West Nile); andTG21.

Other drugs for treatment of influenza and/or hepatitis that areavailable for potential administration in combination or conjunctionwith the compounds and methods disclosed herein include, withoutlimitation:

TABLE 1 Hepatitis and influenza drugs Branded Name Generic Name ApprovedIndications PEGASYS ® PEGinterferon Hepatitis C, (Genentech, alfa-2aHepatitis B South San Francisco, California) PEGINTRON ® PEGinterferonHepatitis C (Merck, alfa-2b Whitehouse Station, New Jersey) COPEGUS ®Ribavirin Hepatitis C (Roche Pharmaceuticals, Nutley, New Jersey)REBETOL ® Ribavirin Hepatitis C (Schering Plough, Kenilworth, NewJersey) — Ribavirin Hepatitis C TAMIFLU ® Oseltamivir Influenza A, B, C(Roche Pharmaceuticals, Nutley, New Jersey) RELENZA ® ZanamivirInfluenza A, B, C (GlaxoSmithKline, London, UK) — Amantadine Influenza A— Rimantadine Influenza A

These agents can be incorporated as part of the same pharmaceuticalcomposition or can be administered separately from the compounds of thedisclosure, either concurrently or in accordance with another treatmentschedule.

The compounds and methods disclosed herein can be additive orsynergistic with other compounds and methods to enable vaccinedevelopment. By virtue of their antiviral and immune enhancingproperties, the compounds can be used to affect a prophylactic ortherapeutic vaccination. The compounds need not be administeredsimultaneously or in combination with other vaccine components to beeffective. The vaccine applications of the compounds are not limited tothe prevention or treatment of virus infection but can encompass alltherapeutic and prophylactic vaccine applications due to the generalnature of the immune response elicited by the compounds.

As is understood by one of ordinary skill in the art, vaccines can beagainst viruses, bacterial infections, cancers, etc. and can include oneor more of, without limitation, a live attenuated vaccine (LAIV), aninactivated vaccine (IIV; killed virus vaccine), a subunit (splitvaccine); a sub-virion vaccine; a purified protein vaccine; or a DNAvaccine. Appropriate adjuvants include one or more of, withoutlimitation, water in oil emulsions, oil in water emulsions, non-ioniccopolymer adjuvants, e.g., CRL 1005 (Optivax™; Vaxcel Inc., Norcross,Ga.), aluminum phosphate, aluminum hydroxide, aqueous suspensions ofaluminum and magnesium hydroxides, bacterial endotoxins,polynucleotides, polyelectrolytes, lipophilic adjuvants and syntheticmuramyl dipeptide (norMDP) analogs such asN-acetyl-nor-muranyl-L-alanyl-D-isoglutamine,N-acetyl-muranyl-(6-O-stearoyl)-L-alanyl-D-isoglutamine orN-Glycol-muranyl-LalphaAbu-D-isoglutamine (Ciba-Geigy Ltd.).

The compounds disclosed herein may be added to other adjuvant compoundsor formulations with adjuvanting properties in order to expand orenhance the immune response to a vaccine. Adjuvants or formulations thatmay be combined with the disclosed compounds include but are not limitedto: squalene, squalene emulsions, tocopherol, tocopherol emulsions,liposomes, virosomes, polyoxidonium, flagellin, Glucopyranosyl LipidAdjuvant, Glucopyranosyl Lipid Adjuvant-stable emulsion,polyinosinic-polycytidylic acid, Covaccine, IC31, Inulin, JVRS-100,monatide, complete Freund's adjuvant, incomplete Freund's adjuvant,viral RNA, bacterial DNA, bacterial RNA, monophosphoryl Lipid A,lipopolysaccharide, Monatide ISA 720, Pluronic F68, Pluronic® L121 (BASFCorporation, Mount Olive, N.J.), Tween® 20 (Sigma-Aldrich), Tween® 80,MF59® (Novartis, Basel, Switzerland), AddaVax™ (InvivoGen, San Diego,Calif.) Span85, DETOX®, lecithin, soy lecithin, egg lecithin,phosphatidylcholine, SB62, AS03, CpG oligodeoxynucleotide, QS21,saponin, AS02, Ceteareth-12, Pluronic®L35 (BASF Corp., New Jersey),Pluronic®L141, Arlacel® (Uniqema Americas, LLC, Wilmington, Del.),paraffin, CoVaccine HT, sucrose fatty acid sulfate ester, SPT, SAF,Provax® (SPX Corporation, Charlotte, N.C.), Brij®98 (Uniqema AmericasLLC, Wilmington, Del.), Brij®30, Castor oil and derivatives, coconut oiland derivatives, corn oil and derivatives, cottonseed oil andderivatives, evening primrose oil and derivatives, fish oil andderivatives, jojoba oil and derivatives, lard oil and derivatives,linseed oil and derivatives, olive oil and derivatives, peanut oil andderivatives, safflower oil and derivatives, sesame oil and derivatives,soybean oil and derivatives, sunflower oil and derivatives, wheatgermoil and derivatives, mineral oil and derivatives,N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, Myverol,TiterMax® Classic (Titermax USA, Inc., Norcross, Ga.), TiterMax® Gold,mannide monooleate, single stranded DNA, double stranded DNA, singlestranded RNA, double stranded RNA, Aluminum salts, aluminum phosphate,aluminum hydroxide, aluminum potassium sulfate, alhydrogel, ISCOM(s)™,cholera toxin, cholera toxin B subunit, Dimethyldioctadecylammoniumbromide, interleukin-12, Etx-B subunit, LTK63, Ribi adjuvant,corynebacterium-derived P40, AS02, AS04, muramyl dipeptide, CRL1005,monatide ISA51, adamantylamide dipeptide, VSA-3, polygen, Bay R1005,Theramide (Vaxio, Ann Arbor, Mich.), stearyl tyrosine, Specol,Algammulin, Avridine™ (Sigma-Aldrich), calcium phosphate gel, DOC/Alumcomplex, Gamma inulin, Gerbu, granulocyte-colony stimulating factor,N-acetylglucosaminyl-(b1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine,interferon gamma, interleukin-1beta, interleukin-2, interleukin-7,sclavo peptide, rehydragel LV, Rehydragel HPA, Loxoribine, MTP-PEliposomes, murametide, Murapamitine, Polymethyl methacrylate, SPT,Quil-A, RC529, LT(R92G), amorphous aluminum hydroxyphosphate sulfate,imiquimod, resimiquimod, AF03, Abisco-100, Albumin-heparin, B7-2, DHEA,SAF-1, threonyl muramyl dipeptide, bupivacaine, interleukin-15,Matrix-S.

The pharmaceutical composition comprising a compound of the disclosurecan be formulated in a variety of forms; e.g., as a liquid, gel,lyophilized, or as a compressed solid. The preferred form will dependupon the particular indication being treated and discernible by one ofordinary skill in the art. In one embodiment, the disclosed RIG-Iagonists include formulations for oral delivery that can besmall-molecule drugs that employ straightforward medicinal chemistryprocesses.

The administration of the formulations of the present disclosure can beperformed in a variety of ways, including, but not limited to, orally,subcutaneously, intravenously, intracerebrally, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,intrathecally, vaginally, rectally, intraocularly, or in any otheracceptable manner. The formulations can be administered continuously byinfusion, although bolus injection is acceptable, using techniques knownin the art, such as pumps (e.g., subcutaneous osmotic pumps) orimplantation. In some instances the formulations can be directly appliedas a solution or spray.

An example of a pharmaceutical composition is a solution designed forparenteral administration. Although in many cases pharmaceuticalsolution formulations are provided in liquid form, appropriate forimmediate use, such parenteral formulations can also be provided infrozen or in lyophilized form. In the former case, the composition mustbe thawed prior to use. The latter form is often used to enhance thestability of the active compound contained in the composition under awider variety of storage conditions, as it is recognized by those ofordinary skill in the art that lyophilized preparations are generallymore stable than their liquid counterparts. Such lyophilizedpreparations are reconstituted prior to use by the addition of one ormore suitable pharmaceutically acceptable diluents such as, withoutlimitation, sterile water for injection or sterile physiological salinesolution.

Parenterals can be prepared for storage as lyophilized formulations oraqueous solutions by mixing, as appropriate, the compound having thedesired degree of purity with one or more pharmaceutically acceptablecarriers, excipients or stabilizers typically employed in the art (allof which are termed “excipients”), for example buffering agents,stabilizing agents, preservatives, isotonifiers, non-ionic detergents,antioxidants and/or other miscellaneous additives.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They are typically present at a concentrationranging from 2 mM to 50 mM. Suitable buffering agents for use with thepresent disclosure include both organic and inorganic acids and saltsthereof such as citrate buffers (e.g., monosodium citrate-disodiumcitrate mixture, citric acid-trisodium citrate mixture, citricacid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additional possibilities are phosphatebuffers, histidine buffers and trimethylamine salts such as Tris.

Preservatives can be added to retard microbial growth, and are typicallyadded in amounts of 0.2%-1% (w/v). Suitable preservatives for use withthe present disclosure include, without limitation, phenol, benzylalcohol, meta-cresol, methyl paraben, propyl paraben,octadecyldimethylbenzyl ammonium chloride, benzalkonium halides (e.g.,benzalkonium chloride, bromide or iodide), hexamethonium chloride, alkylparabens such as methyl or propyl paraben, catechol, resorcinol,cyclohexanol and 3-pentanol.

Isotonicifiers can be added to ensure isotonicity of liquid compositionsand include, without limitation, polyhydric sugar alcohols, preferablytrihydric or higher sugar alcohols, such as glycerin, erythritol,arabitol, xylitol, sorbitol and mannitol. Polyhydric alcohols can bepresent in an amount between 0.1% and 25% by weight, typically 1% to 5%,taking into account the relative amounts of the other ingredients.

Stabilizers refer to a broad category of excipients which can range infunction from a bulking agent to an additive which solubilizes thetherapeutic agent or helps to prevent denaturation or adherence to thecontainer wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, ornithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur-containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol,alpha-monothioglycerol and sodium thiosulfate; low molecular weightpolypeptides (i.e., <10 residues); proteins such as human serum albumin,bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymerssuch as polyvinylpyrrolidone; monosaccharides such as xylose, mannose,fructose and glucose; disaccharides such as lactose, maltose andsucrose; trisaccharides such as raffinose, and polysaccharides such asdextran. Stabilizers are typically present in the range of from 0.1 to10,000 parts by weight based on the active compound weight.

Additional miscellaneous excipients include fillers (e.g., starch),chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid,methionine, vitamin E) and cosolvents.

The active ingredient can also be entrapped in microcapsules prepared,for example, by coascervation techniques or by interfacialpolymerization, for example hydroxymethylcellulose, gelatin orpoly-(methylmethacylate) microcapsules, in colloidal drug deliverysystems (for example liposomes, albumin microspheres, microemulsions,nano-particles and nanocapsules) or in macroemulsions. Such techniquesare disclosed in Remington, The Science and Practice of Pharmacy,21^(st) Ed., published by Lippincott Williams & Wilkins, A WoltersKluwer Company, 2005, the teachings of which are incorporated byreference herein.

Parenteral formulations to be used for in vivo administration generallyare sterile. This is readily accomplished, for example, by filtrationthrough sterile filtration membranes.

Suitable examples of sustained-release preparations includesemi-permeable matrices of solid hydrophobic polymers containing thecompound or composition, the matrices having a suitable form such as afilm or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) orpoly(vinylalcohol)), polylactides, copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the PROLEASE® technology(Alkermes, Cambridge, Mass.) or LUPRON DEPOT® (injectable microspherescomposed of lactic acid-glycolic acid copolymer and leuprolide acetate;Abbott Laboratories, Abbott Park, Ill.), and poly-D-(−)-3-hydroxybutyricacid. While polymers such as ethylene-vinyl acetate and lacticacid-glycolic acid enable release of molecules for long periods such asup to or over 100 days, certain hydrogels release compounds for shortertime periods.

Oral administration of the compounds and compositions is one intendedpractice of the disclosure. For oral administration, the pharmaceuticalcomposition can be in solid or liquid form, e.g., in the form of acapsule, tablet, powder, granule, suspension, emulsion or solution. Thepharmaceutical composition is preferably made in the form of a dosageunit containing a given amount of the active ingredient. A suitabledaily dose for a human or other vertebrate can vary widely depending onthe condition of the patient and other factors, but can be determined bypersons of ordinary skill in the art using routine methods.

In solid dosage forms, the active compound can be admixed with at leastone inert diluent such as sucrose, lactose, or starch. Such dosage formscan also comprise, as is normal practice, additional substances, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets and pills, the dosage forms can also comprise buffering agents.Tablets and pills can additionally be prepared with enteric coatings.

The compounds or compositions can be admixed with adjuvants such aslactose, sucrose, starch powder, cellulose esters of alkanoic acids,stearic acid, talc, magnesium stearate, magnesium oxide, sodium andcalcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodiumalginate, polyvinyl-pyrrolidine, and/or polyvinyl alcohol, and tabletedor encapsulated for conventional administration. Alternatively, they canbe dissolved in saline, water, polyethylene glycol, propylene glycol,ethanol, oils (such as corn oil, peanut oil, cottonseed oil or sesameoil), tragacanth gum, and/or various buffers. Other adjuvants and modesof administration are known in the pharmaceutical art. The carrier ordiluent can include time delay material, such as glyceryl monostearateor glyceryl distearate alone or with a wax, or other materials known inthe art.

The present disclosure further includes the use and application of thecompounds, compositions and methods herein in vitro in a number ofapplications including but not limited to developing therapies andvaccines against viral infections, research in modulation of the innateimmune response in eukaryotic cells, etc. The compounds, compositionsand methods of the present disclosure can also be used in animal models.The results of such in vitro and animal in vivo uses of the compounds,compositions and methods of the present disclosure can, for example,inform their in vivo use in humans, or they can be valuable independentof any human therapeutic or prophylactic use.

EXAMPLES

The Examples below describe the antiviral and pharmacological propertiesof compounds within the disclosed compounds. The Examples are includedto demonstrate particular embodiments of the disclosure. It should beappreciated by those of ordinary skill in the art that the techniquesdisclosed in the Examples represent techniques and compositionsdiscovered by the inventors to function well in the practice of thedisclosure, and thus can be considered to constitute preferred modes forits practice. However, those of ordinary skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe disclosure. For example, the Examples below provide in vitro methodsfor testing the compounds of the disclosure. Other in vitro virusinfection models include but are not limited to flaviviruses such asbovine diarrheal virus, West Nile Virus, and GBV-C virus, other RNAviruses such as respiratory syncytial virus, and the HCV repliconsystems. Furthermore, any appropriate cultured cell competent for viralreplication can be utilized in the antiviral assays.

Example 1 Biological Activity of KIN1000

Luciferase assay to identify active compounds. Cultured human cells thatwere stably transfected with a luciferase reporter gene coupled with aRIG-I signaling pathway responsive promoter (IFNβ, ISG56, or ISG54promoter) were seeded and allowed to grow overnight. The compound“KIN1000” was then added and cells were grown in the presence of KIN1000for 18-20 hours. Steady-Glo luciferase substrate (Promega) was added andluminescence was read on a luminometer (Berthold).

FIG. 1A shows that KIN1000 as described herein was validated bydemonstrating dose-dependent induction of the luciferase reporter genecoupled to the promoters for IFNβ (“IFNβ-LUC,” left), ISG56(“ISG56-LUC,” center), and ISG54 (“ISG54-LUC,” right). Additionally,KIN1000 did not induce a nonspecific promoter (β-actin-LUC, FIG. 1B).

MTS assay to determine cytotoxicity. Cultured human HeLa cells weretreated with increasing amounts of compound or equivalent amounts ofDMSO diluted in media for 48 hours to see their effect on cellviability. The proportion of viable cells was calculated using a cellviability assay that measures conversion of a tetrazolium compound[3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; or MTS] to a colored formazan compound in live cells.

The conversion of MTS to formazan was detected in a 96-well microtiterplate reader, and the resulting optical densities plotted directly toestimate cell viability. Cell Titer One (Promega) was the one-stepreagent used, as manufacturer's protocol suggested, and cells wereincubated for three hours in the presence of reagent before opticaldensity (O.D.) reading was done. Compounds were diluted to finalconcentrations of 0, 1, 5, 10, and 20 μM in media containing 0.5% DMSO.Negative control wells contained no compound, and positive control forcytotoxicity was examined using 10% DMSO. Each KIN1000 concentration andcontrol was done in triplicate wells. KIN1000 showed no cytotoxicity tomultiple cell types (MTS assay, FIG. 1C).

Immunofluorescent cytochemistry assay to determine IRF-3 activation andtranslocation to the nucleus. The induction of ISG expression mediatedby RIG-I is conferred by phosphorylation, dimerization, and nucleartranslocation of the IRF-3 transcription factor. Cultured human HeLacells were treated with increasing amounts of compound or equivalentamounts of DMSO diluted in media for 20 hours. Positive control wellswere infected with 100 HA/mL Sendai virus for an equivalent time period.IRF-3 was detected using polyclonal rabbit serum specific to IRF-3 and asecondary antibody conjugated to DyLight® (Pierce Biotechnology, Inc.,Rockford, Ill.) 488. KIN1000 shows a dose dependent increase innuclear-cytoplasmic difference for IRF-3 (FIG. 2A).

Immunofluorescent cytochemistry assay to determine NFκB activation. Theinnate immune response dependent on RIG-I also activates the NFκBtranscription factor and thus increases nuclear levels. Cultured humanHeLa cells were treated with increasing amounts of compound orequivalent amounts of DMSO diluted in media for 20 hours. Positivecontrol wells were infected with 100 HA/mL Sendai virus for anequivalent time period. NFκB was detected using monoclonal mouseantibody specific to the p65 subunit of NFκB and a secondary antibodyconjugated to DyLight 488.

Quantification of immunofluorescent assays. 96-well plates containingcultured human cells treated with compound and stained for either IRF-3or NFκB were scanned and quantified using the ArrayScan® instrument andsoftware (Cellomics, Inc., Pittsburgh, Pa.). Activation of transcriptionfactor was evidenced by increased nuclear intensity normalized forcytoplasmic intensity, or nuclear-cytoplasmic difference. KIN1000 showsa dose dependent increase in nuclear-cytoplasmic difference for NFκB(FIG. 2B).

Other compounds as described herein likewise can be evaluated by themethods described in this example, and other cell types can also beused.

Example 2 Antiviral Activity of KIN1000 against Influenza WSN Strain

MRC5 cells were treated with increasing amounts of KIN1000 12-24 hoursprior to infection by influenza virus. The number of infected cells 24hours after introduction of virus was then quantified by animmunofluorescent assay of viral protein in cells. The KIN1000 compounddisclosed herein demonstrated efficient activity against influenza virusstrain WSN. FIG. 3 shows that MRC5 cells treated with increasing amountsof KIN1000 showed a dose-dependent decrease in infection by influenzavirus.

Example 3 Ex Vivo Immune Stimulatory Activity of KIN1000

The activity of KIN1000 in primary immune cells was assayed to determinewhether KIN1000 stimulates immune responses. Cultured human primarydendritic cells were treated with 0, 1, or 10 μM of KIN1000 for 24hours. Supernatant from treated wells was isolated and tested for levelsof cytokine protein. Cytokines were detected using specific antibodiesconjugated to magnetic beads and a secondary antibody that reacts withStreptavidin/Phycoerythrin to produce a fluorescent signal. The boundbeads were detected and quantified using the Magpix® (Luminex Corp.,Austin, Tex.) instrument, although similar techniques as are known inthe art may be used to measure fluorescent protein production, such asfor example an ELISA.

KIN1000 was shown to induce expression of the chemokines IL-8, MCP-1,MIP-1α and MIP-1β by dendritic cells (FIGS. 4A-4D).

Other cells from which cytokine secretion can be measured include, forexample but without limitation, human peripheral blood mononuclearcells, human macrophages, mouse macrophages, mouse splenocytes, ratthymocytes, and rat splenocytes.

Example 4 Antiviral Activity and Pharmacological Properties usingStructure-Activity Relationship (SAR) Studies

This Example describes optimization of compounds for antiviral action.First, a small analog derivative set is used to define a structuralclass. The active analogs that are identified in this first stage arethen used to define a subset of structural classes of interest forfurther optimization (Stage 2).

Stage 2, derivative expansion. Stage 2 focuses on creating structuraldiversity and evaluating core variants. Structural derivatives aretested for biological activity in the IRF-3 translocation assay,antiviral activity, and cytotoxicity in one or more cell lines orperipheral blood mononuclear cells. Optimized molecules that showimproved efficacy and low cytotoxicity are further characterized byadditional measures of in vitro toxicology and absorption, distribution,metabolism, and elimination (ADME). Their mechanism of action andbreadth of antiviral activity are also studied.

Chemical design in SAR studies. To design analog structures, thedrug-like properties, metabolic lability, and toxic potential of thelead compounds are analyzed. Drug-like properties, as measured byLipinski's Rules, and related physiochemical properties are primaryindicators of bioavailability. Structural features that suggestmetabolic and toxicological liabilities may indicate limited stability,reduced half-life, reactive intermediates, or idiosyncratic toxicity andwill therefore be removed. A 5- to 10-compound analog set is constructedto remove or alter chemically reactive or metabolically susceptiblestructural features, thereby developing a preliminary SAR.

Compounds are tested for in vitro antiviral activity against HCV 2A,respiratory syncytial virus, dengue virus type 2, and influenza A virusstrains. Viral protein and RNA levels are assessed following drugtreatment using the assays described herein.

Following several iterative rounds of SAR, compounds are selected forcharacterization of their in vitro toxicological and ADME properties andfor further mechanistic study. The SAR studies are designed to providelead compounds with picomolar to nanomolar potency, which is adequate tosupport preclinical development.

In vitro pharmacology. In vitro pharmacology studies are performed tomeasure performance of the most promising analogs in one or more assaysof intestinal permeability, metabolic stability and toxicity. Key invitro characterization studies can include plasma protein binding;serum, plasma, and whole-blood stability in human and model organisms;intestinal permeability; intrinsic clearance; human Ether-à-go-go (hERG)channel inhibition; and genotoxicity.

For each analog, an HPLC- and/or HPLC-mass spectrometry-based analyticalmethod is used to evaluate drug and metabolite concentrations in varioustest systems. Although the specific analytical method is optimized foreach molecule, reverse-phase chromatography can be used alone or incombination with quadrupole mass spectrometry to characterize theidentity and purity of several of the lead molecules. Initially, drugstability over time in increasing concentrations of serum, plasma, andwhole blood from mammalian species (such as mouse, cynomolgus macaque,and human) is evaluated by HPLC, and a half-life is determined.

Prominent metabolites characterized by mass spectrometry. Human plasmaprotein binding are evaluated by partition analysis using equilibriumdialysis. For intestinal permeability modeling, apical-to-basolateralflux is assessed in the human epithelial cell line TC7. Hepaticclearance is estimated for a subset of the most promising analogs bymeasuring the rate of disappearance of the parent compound duringincubation in human liver microsomes. As above, specific metabolites canbe isolated and characterized.

In vitro toxicology. In vitro toxicology studies are performed toevaluate the potential cardiac and genetic toxicity of lead analogs.Automated patch-clamp is used to assess the impact of each compound onhERG channel currents in a recombinant Chinese hamster ovary (CHO) cellline transgenically expressing the human Kv11.1 gene. Concentrations upto the lesser of 30 times the maximum serum concentration or the limitof solubility of each compound are evaluated in order to determine an1050 for the molecule on the hERG channel. A subset of compounds isevaluated over a range of concentrations for their ability to inducemutation reversion in Salmonella typhimurium strains TA98 and TA100 orto promote micronucleus formation in CHO cells in culture.

Example 5 Activation of Gene Expression by KIN1000 and DerivativeCompounds

Gene expression in HeLa cells. Cultured human cells were treated with 20μM, 10 μM, 5 μM of compound or a DMSO control and incubated for up to 24hours. Cells were harvested and RNA was isolated using the QIAshreddercolumns and RNeasy Mini Kit (Qiagen) according to manufacturerinstructions. Reverse transcription was performed and the cDNA templatewas used for quantitative real-time PCR. PCR reactions were performedusing commercially available, validated TaqMan gene expression assays(Applied Biosystems/Life Technologies) according to manufacturerinstructions. Gene expression levels were measured using a relativeexpression analysis (ΔΔCt).

Gene expression in PH5CH8 cells. Cultured human cells were treated with10 uM, 5 uM, 1 uM or a DMSO control and incubated for up to 24 hours.Cells were harvested and RNA was isolated using the QIAshredder columnsand RNeasy Mini Kit (Qiagen) according to manufacturer instructions.Reverse transcription was performed and the cDNA template was used forquantitative real-time PCR. PCR reactions were performed usingcommercially available, validated TaqMan gene expression assays (AppliedBiosystems/Life Technologies) according to manufacturer instructions.Gene expression levels were measured using a relative expressionanalysis (ΔΔCt).

Gene expression in HUVEC primary cells. Cells were thawed and seeded in6-well plates at 2.4×10⁴ cells per well and allowed to grow to 80%confluence, typically 5 days in culture with fresh media replaced every48 hours. Compound was added at 10 μM, 1 μM or a DMSO control andincubated for up to 24 hours. Cells were harvested and RNA was isolatedusing the QIAshredder columns and RNeasy Mini Kit (Qiagen) according tomanufacturer instructions. Reverse transcription was performed and thecDNA template was used for quantitative real-time PCR. PCR reactionswere performed using commercially available, validated TaqMan geneexpression assays (Applied Biosystems/Life Technologies) according tomanufacturer instructions. Gene expression levels were measured using arelative expression analysis (ΔΔCt).

FIGS. 5A-5F show induction of gene expression by KIN1000 and itsderivative compound KIN1148. FIG. 5A shows gene expression levels ofIFIT2 and FIG. 5B shows OAS1 in HeLa cells over time from 4-24 hourspost treatment with 10 μM KIN1000 (grey) or 10 μM KIN1148 (black). FIG.5C shows gene expression levels of IFIT2 in PH5CH8 cells (left) treatedwith KIN1000 (solid grey bars) or KIN1148 (solid black bars), and inHeLa cells (right) treated with KIN1000 (grey striped bars) or KIN1148(black checked bars). In each test group, the three vertical barsrepresent 5, 10, and 20 μM compound (KIN1000 or KIN1148), respectively.FIG. 5D shows gene expression levels of IFIT2, FIG. 5E shows OAS1, andFIG. 5F shows MxA in primary HUVEC cells that were treated with 1 μMKIN1000 (grey) or 1 μM KIN1148 (black). The difference in axis scalingdemonstrates that compounds are more active in a primary cell type.These data demonstrate that compounds are active in cells by inducingresponsive gene expression.

Gene expression can be similarly assayed in cell types that include,without limitation: primary blood mononuclear cells, human macrophages,THP-1 cells, Huh 7 cells, A549 cells, MRC5 cells, rat splenocytes, ratthymocytes, mouse macrophages, mouse splenocytes, and mouse thymocytes.Expression of other genes of interest can be assayed as describedherein.

Example 6 Antiviral Activity of KIN1000 against Various Viruses

Antiviral action in cell culture infection models. To furthercharacterize the breadth of antiviral activity of optimized molecules,cell culture infection models are used to analyze different viruses,including but not limited to different strains of influenza virus, HCV,Dengue virus, RSV, and West Nile virus (WNV), an emerging public healthconcern. The studies include treating cells with compound 2-24 hoursprior to infection or treating cells up to 8 hours after infection.Virus production and cellular ISG expression are assessed over a timecourse to analyze antiviral effects of representative compounds fromlead structural classes. IFNβ treatment is used as a positive control.

Virus production is measured by focus-forming or plaque assay. Inparallel experiments, viral RNA and cellular ISG expression are measuredby qPCR and immunoblot analyses. These experiments are designed tovalidate compound signaling actions during virus infection, and assesscompound actions to direct innate immune antiviral programs againstvarious strains of viruses and in the setting of virus countermeasures.Detailed dose-response analyses of each compound are conducted in eachvirus infection system to determine the effective dose that suppressesvirus production by 50% (IC50) and 90% (IC90) as compared with controlcells for both the pre-treatment and post-treatment infection models.

TABLE 2 Virus systems and study design for antiviral analysis of leadcompounds Virus Virus Strain Study Design HCV H77 (genotype 1a) AssaysJFH1 (genotype 2a) Plaque or focus forming assays FLU High pathogenicityin mice (infectious virus) A/PR/8/34 (H1N1 qPCR (RNA levels)mouse-adapted virus) Immunoblot and ELISA A/WSN/33 (H1N1 (proteinlevels) mouse-adapted Study Design neurovirulent virus) Compoundtreatment of cells Low pathogenicity in mice pre- and post-infectionA/Texas/36/91 (H1N1 Determine EC₅₀ and EC₉₀ circulating virus)Inhibition of viral life cycle A/Udorn/72 (H3N2) WNV TX02 (lineage 1)MAD78 (lineage 2)

Example 7 Activity of KIN1000 and Derivative Compounds againstRespiratory Syncytial Virus

HeLa cells were seeded the previous day in 6-well plates at 4×10⁵ cellsper well. The next day, the media was replaced with RSV in media withoutFBS at an MOI of 0.1. Virus binding occurred at 37° C. for 2 hours.After 2 hours the cells were washed with warm complete media andreplaced with media containing drug at varying concentrations of 10 μM,5 μM, 1 μM or a DMSO control. Cells were placed in a 37° C. incubatorfor 48 hours.

For virus detection and titration, HeLa cells were seeded in 96-wellplates at 8×10³ cells per well 24 hrs prior to collecting virussupernatant. After the 48 hour incubation period, the virus supernatantfrom the infected plate was harvested and used to infect these cells ata 1/10 final dilution. Cells were placed in a 37° C. incubator for 24hours.

24 hours after infection, cells were washed twice with PBS and fixedwith methanol/acetone solution. After fixing the cells were washed twicewith PBS and replaced with blocking buffer (10% horse serum, 1 g/mL BSAand 0.1% Triton-100× in PBS) for 1 hour. The blocking buffer is replacedwith binding buffer containing a 1/2000 dilution of primary antibody for2 hours at room temperature. The primary antibody was a mouse monoclonalantibody against RSV. The cells were washed twice with PBS and replacedwith binding buffer containing 1/3000 dilution of the Alexa Fluor-488goat anti-mouse secondary antibody and a Hoechst nuclear stain for 1hour at room temperature. The cells were washed twice with PBS and PBSis added to all wells. The 96-well plate is sealed and fluorescenceactivity associated with virus infectivity was determined byimmunofluorescent assay using the Array Scan instrument(Thermo-Fischer).

FIGS. 6A-6B show experiments performed using the protocol of theExample, demonstrating the antiviral activity of KIN1000 and KIN1148against respiratory syncytial virus. FIG. 6A shows that HeLa cellstreated with increasing amount of KIN1000 and KIN1148 showeddose-dependent decrease in infection by RSV. FIG. 6B shows that KIN1148showed antiviral activity against RSV when drug is added up to 24 hoursprior to infection.

Treatment with compounds prior to infection. In variations of thismethod, the compounds are added at varying time points prior toinfection with virus. Virus detection and titration is conducted asdescribed.

Analog testing and SAR studies. Antiviral activity against RSV was usedas a criterion to measure activity of structural derivatives of KIN1000.Table 3 shows select structural derivatives of KIN1000 that demonstratedantiviral activity against RSV. Compared to KIN1000 parent compound,these analogs showed varying levels of antiviral activity against RSV.+++=greater than 70% inhibition of infection, ++=greater than 50%inhibition, +=greater than 30% inhibition, −=less than 30% inhibition.

TABLE 3

KIN R3 R1 R2 10 uM 5 uM 1 uM 1000

H ++ + − 1014

H + − − 1034

H − + − 1069

H H ++ + − 1072

H H ++ − − 1075

+ − − 1148

H H +++ ++ − 1169

H +++ ++ − 1170

H ++ − − 1203

H H +++ − −

Example 8 Activity of KIN1000 and Derivative Compounds against InfluenzaA/Udorn/72 Virus

Influenza A/Udorn/72 infection of H292 cells. 0.2×10⁶ H292 cells inRPMI1640+10% FCS were treated with 2 μM KIN1148 in a final concentrationof 0.5% DMSO for 6 hours. Compound-containing media was aspirated andreplaced with 1× MEM containing A/Udorn/72 at an MOI of 0.1 and placedat 37° C. in a CO² incubator. Two hours post infection, virus-containingmedia was aspirated and replaced with 1× MEM containing 1 ug/mLTPCK-treated Trypsin, 2 μM KIN1148, 0.5% DMSO. Cells were placed in 37°C. CO₂ incubator for 18 hours. After 20 hours post-infection, virussupernatants were collected and titred on MDCK cells.

Influenza A/Udorn/72 infection of HEK293 cells. 5×10⁵ HEK293 cells wereinfected with A/Udorn/72 at an MOI of 0.2 in 1× MEM. After 2 hourspost-infection, virus-containing media was aspirated and replaced with1× MEM containing 1 μg/mL TPCK-treated Trypsin, 10 μM KIN1148, 0.5%DMSO. Cells were returned to 37° C., CO₂ incubator for 18 hours. After20 hours post-infection, virus supernatants were collected and titred onMDCK cells.

Titre in MDCK cells. 10 μL of infected supernatant was added to 2×10⁶MDCK cells in the presence of 2 μg/mL TPCK-trypsin and placed in a 37°C. CO₂ incubator. After 8 hours, supernatant was removed and cells werefixed and stained with FITC-conjugated antibody specific for InfluenzaNP protein. Number of foci was quantitated using the ArrayScaninstrument and software (Cellomics).

FIGS. 7A-7B show antiviral activity of KIN1148 against Influenza A virusUdorn/72. H292 cells (FIG. 7A) and HEK293 cells (FIG. 7B) treated with 2uM (H292) or 10 uM (HEK293) of KIN1148 showed decrease in infection byvirus.

Example 9 Activity of KIN1000 and Derivative Compounds against DengueVirus

Huh 7 cells were seeded the previous day in 6-well plates with 4×10⁵cells per well. The next day, the media was replaced with Dengue virustype 2 in media without FBS at an MOI of 0.25. Virus binding occurred at4° C. for 1 hour. After 1 hour the cells were washed with warm completemedia and replaced with media containing KIN1148 at varyingconcentrations of 10 uM, 5 uM, 1 uM or a DMSO control. Cells were placedin a 37° C. incubator for 48 hours.

Titre in Vero cells. Vero cells were seeded in 96-well plates at 8×10³cells per well 24 hrs prior to collecting virus supernatant. After 48hrs, the virus supernatant was harvested and used to infect Vero cellsat a 1/100 final dilution.

24 hrs after infection, Vero cells were washed 2× with PBS and fixedwith methanol/acetone for 15 mins. After fixing the cells were wash 2×with PBS and replaced with blocking buffer for 30-45 mins. The blockingbuffer was replaced with binding buffer containing a 1/2000 dilution ofprimary monoclonal antibody targeting the Envelope protein for 2 hrs.After 2 hrs, the cells were washed 2× with PBS and replaced with bindingbuffer containing 1/3000 dilution of the Alexa Fluor-488 goat anti-mousesecondary antibody and a Hoechst nuclear stain for 45 mins. After 45mins cells were washed 2× with PBS and PBS was added to all the well.The 96-well plate was sealed and fluorescence activity associated withvirus infectivity was determined by IF using the ArrayScan instrumentand software (Cellomics).

FIG. 8 shows the results of experiments performed using the protocol ofthis Example, demonstrating the antiviral activity of KIN1148 againstDengue virus type 2. Huh 7 cells treated with increasing amounts ofKIN1148 showed dose-dependent decrease in infection by virus.

Example 10 Activity of KIN1000 and Derivative Compounds againstHepatitis B Virus

HepAD38 cells (Hep 2 cells expressing a regulated HBV genome) were grownfor 72 hours in the presence of compound (concentrations 1-10 μM in 0.5%DMSO media). HepAD38 cells that do not express HBV were used as anegative control. Following 72 hours of treatment 100 μl of media wasused in an ELISA to measure HBV surface antigen. The amount of HBVsurface antigen produced by the cells was measured in the supernatantsby ELISA commercially available HBV sAg ELISA from Creative Diagnostics,N.J.

FIG. 9 shows the results of experiments performed using the protocol ofthis Example, demonstrating the antiviral activity of KIN1148 againstHepatitis B virus. HepAD38 cells treated with increasing amounts ofKIN1148 showed dose-dependent decrease in supernatant levels of virus.

Example 11 In Vivo Pharmacokinetic, Toxicological, and AntiviralProperties of Optimized Drug Leads in Relevant Preclinical Animal Models

Preclinical pharmacokinetic and tolerability profiling. The in vivopharmacokinetic (PK) profile and tolerability/toxicity of KIN1000 andrelated compounds are evaluated in order to conduct furthercharacterization of their antiviral activity in animal models ofinfluenza virus and WNV infection. Mouse is the chosen test species forthese studies since it is the most commonly used rodent model of WNV andinfluenza.

A reverse-phase, HPLC-MS/MS detection method is used for measuring theconcentration of each compound in mouse plasma. Prior to PK profiling,an initial oral and intravenous formulation for each compound isdeveloped using a limited formulation component screen that is largelyfocused on maximizing aqueous solubility and stability over a smallnumber of storage conditions. Any of the analytical methods as are knownin the art can be used to measure formulation performance. A formulationis developed for each compound following a three tiered strategy:

-   Tier 1: pH (pH 3 to 9), buffer, and osmolality adjustment-   Tier 2: addition of ethanol (<10%), propylene glycol (<40%), or    polyethylene glycol (PEG) 300 or 400 (<60%) co-solvents to enhance    solubility-   Tier 3: addition of N-N-dimethylacetamide (DMA, <30%),    N-methyl-2-pyrrolidone (NMP, <20%), and/or dimethyl sulfoxide (DMSO,    <20%) co-solvents or the cyclodextrins (<40%) as needed to further    improve solubility.

For compounds that demonstrate adequate performance in in vitroantiviral, mechanistic, ADME, and toxicology studies, a preliminarymouse PK study is performed. See Table 3. Each compound is administeredas a single dose to animals by oral gavage (<10 ml/kg) or i.v. bolusinjection (<5 ml/kg) after an overnight fast. Multiple animals are dosedfor each dosing group such that 3 animals can be sampled at each timepoint. Blood samples are collected by retro-orbital sinus prior todosing and at 5, 15, and 30 minutes, and 1, 2, 4, 8, and 24 hourspost-dosing. Drug concentrations are measured according to thepreviously developed bioanalytical method. Pharmacokinetic parametersare evaluated using the WinNonlin software.

TABLE 4 Experimental Route of Study design administration Outcomes MousePK Single dose IV and Oral Oral bioavailability, pharmacokineticC_(max), t_(1/2), Cl, V_(d), study AUC_(0-24, 0-∞) Mouse Phase 1: OralMTD, acute toxicity, tolerability ascending dose hematology, serumtolerability and chemistry, gross MTD pathology determination; Phase 2:placebo controlled 7-day toxicity at MTD

Based upon performance in exploratory PK studies, compounds are furtherevaluated for preliminary tolerability and toxicity in mice prior totheir characterization in antiviral models. Tolerability studies areperformed in two stages: an initial dose escalation stage (up to 5doses, each separated by a 5-day washout period) to determine themaximum tolerable dose (MTD, Phase 1), followed by seven dailyadministrations of the MTD to evaluate acute toxicity (Stage 2). SeeTable 3. All doses are administered by oral gavage. In an exemplaryexperiment, five animals of each sex are placed on-study in stage 1 and15 animals per sex per dosing group in Stage 2. Study endpoints includea determination of the MTD, physical examination, clinical observations,hematology, serum chemistry and animal bodyweights. Gross pathology isperformed on all animals whether found dead, euthanized in extremis, orat the intended conclusion of the experiment. The toxicology studies areprimarily exploratory in nature and intended to identify earlytoxicological endpoints, and drive selection of lead candidates forantiviral animal models.

TABLE 5 In vivo studies of compound actions against WNV and influenzavirus Exemplary No. Experiment Analysis Goal of Mice* Effective Viralburden Define in 238 compound dose analysis in vivo EC₅₀ determinationserum and EC₉₀ Viral Time to moribund Define compound 739 pathogenesisstate, clinical action toward study 1: scoring limiting EC₅₀ and EC₉₀for pathologic signs of viral pathogenesis Treatment infection Viralpathogenesis Viral burden Define compound 1056 study 2: analysis inserum action toward EC₅₀ and EC₉₀ and various target limiting virustreatment and time organs replication and course analysis spread Viralpathogenesis Time to moribund Define compound 370 study 3: state,clinical scoring action toward (neuroinvasion for pathologic signs oflimiting viral model) infection pathogenesis in EC₅₀ and EC₉₀ the CNStreatment *Numbers reflect an average of at least two iterations of eachexperiment

Evaluation of antiviral properties and immune protection using mouseinfection models. Optimized compounds are selected based on compoundpharmacokinetic, antiviral, and innate immune actions for furtherevaluation in preclinical mouse models of infection. See Table 4. Innateimmune actions of the compounds are measured, and their ability toprotect mice from WNV and influenza virus challenge is assessed. For theWNV infection model, subcutaneous footpad infection of wild-type C57BI/6mice with the virulent lineage 1 strain of WNV (WNV-TX) are performed.Non-surgical tracheal instillation is performed for influenza virusstrains A/PR/8/34, A/WSN/33, and A/Udorn/72.

The influenza virus strains used for certain experiments are of twodifferent subtypes (H1N1 and H3N2) and exhibit varying pathogenicproperties and clinical presentations in C57BI/6 mice. Mice aremonitored for morbidity and mortality over a range of challenge doses(such as, 10 to 1,000 pfu of virus) either alone or in combination withcompound treatment beginning 12 hours before or 24 hours after infectionand continuing daily subject to the determined plasma half-life of thedrug. Compound dose-response analysis and infection time course studiesare conducted to evaluate compound efficacy to: 1) limit serum viralload, 2) limit virus replication and spread in target organs, and 3)protect against viral pathogenesis.

For WNV, in addition to serum, viral burden is assessed in lymph nodes,spleen, and brain; for influenza virus, viral burden is assessed inheart, lung, kidney, liver, and brain. Incorporated in the design ofthese experiments is the determination of an effective dose for 50% and90% suppression of serum viral load (ED50 and ED90) by each compoundafter a standard challenge of 100 pfu of WNV-TX or 1,000 pfu ofinfluenza virus. Serum viral loads are determined by qPCR of viral RNAat 24-hour intervals following compound treatment. The compound actionsare tested at the ED50 and ED90 toward limiting WNV pathogenesis in thecerebral nervous system using a WNV neuroinvasion model of infection.

Mice are monitored for morbidity and mortality after standardintracranial challenge of 1 pfu of WNV-MAD, either alone or incombination with compound treatment beginning 24 hours after infection.

Example 12 Antiviral Activity of KIN1000 and Derivative Compounds InVivo

Evaluation of antiviral properties of KIN1000 and derivative compoundsusing mouse infection models. Up to 5 of the most promising compoundswill be selected for further evaluation in preclinical mouse models ofinfection with Influenza and/or respiratory syncytial virus. Table 6lists the nonclinical studies to measure antiviral efficacy of KIN1000and derivative compounds.

TABLE 6 Nonclinical studies to measure drug concentration and antiviralefficacy in vivo Experimental Route No. No. Study design Admi. Cpds.Animals Outcomes Mouse Drug measured Oral/IP ≦5 120 Drug dosing in bloodat 3 concentration dose levels; 2, 8, in blood; HPLC 24 hours postreverse phase treatment Mouse Tracheal Oral/IP ≦5 480 Mortality, viralInfluenza instillation titer in serum/ Model of A/WSN/33 or targetorgans, A/Udorn/72; body temp., drug treatment bodyweight, at 2 doses w/clin. obs., placebo cytokine levels Mouse Tracheal Oral/IP ≦5 240Mortality; RSV instillation bodyweight; Model of RSV A2 Long targetorgan strain; drug viral titer; treatment at 2 innate immune doses w/geneexpression; placebo control markers of inflammation

Mouse influenza model. We will perform non-surgical trachealinstillation of influenza virus strains A/WSN/33 and A/Udorn/72. Theseinfluenza virus strains are two different subtypes (H1N1 and H3N2) andexhibit varying pathogenic properties and clinical presentations inC57B1/6 mice. Lead derivatives of the KIN1000 family of compounds willbe administered daily by oral gavage or IP administration over theentire course of infection (typically 2 weeks) at 2 dose levels plus aplacebo control group. Five animals per sex and per group will beevaluated for endpoints, including but not limited to daily clinicalobservations, mortality, body weight, and body temperature. Threeanimals per sex will be used to measure virus titer in serum, heart,lung, kidney, liver, and brain. Cytokine expression at various timepoints during infection in compound-treated versus control animals willbe assayed.

Mouse RSV model. We will perform non-surgical tracheal instillation ofrespiratory syncytial virus A2 long strain. BALB-c mice will be infectedat a dose of RSV A2 virus that does not cause cytopathic effectsfollowed by daily oral or IP administration of compound at 2 dose levelsor a placebo control for up to 21 days. Mice will be monitored asdescribed above, including inspection for morbidity and mortality, viraltiter in serum and blood, cytokine secretion, increased immune cellpopulations, and innate immune gene expression.

Example 13 Adjuvant Activity of KIN1000 and Derivative Compounds In Vivo

To characterize the breadth of adjuvant activity of KIN1000 and relatedcompounds, in vivo animal models of vaccination and vaccination plusprotection are used. The studies include priming animals including butnot limited to rats and mice with compound alone or in combination withan antigen and then assessing the adjuvant effect.

Adjuvant effect is measured by assays for modified, enhanced immunehumoral and cellular responses. Humoral responses are assessed over timeat discrete times post vaccination and/or boosting by collecting bloodfor sera and determining relative concentrations of antibody classes(IgM, IgG, IgA or IgE) and/or isotypes including IgG1, IgG2a, IgG2b,IgG2c, IgG3 for IgG antibodies. Moreover, affinity and avidity of thegenerated antibodies is also determined. In instances in which thevaccine preparation includes a combination of compound and antigen, theneutralizing activity of the generated antibodies is also determined.

Cellular mediated immune responses induced by the compounds are measuredby established methods in the field including ex vivo stimulation ofperipheral blood mononuclear cells, lymph nodes, splenocytes or othersecondary lymphoid organs with the antigen and measurement of cytokineor chemokine production in the supernatant at several times thereafter.Cytokines measured include Th1 type of cytokines including but notlimited to IFN gamma and TNF alpha, Th2 type cytokines including but notlimited to IL-4, IL-10, IL-5 and IL-13 and Th17 cytokines including butnot limited to IL-17, IL-21 and IL-23. Chemokines elicited by thecompounds are also measured including but not limited to RANTES, IP-10,MIP1a, MIP1b, and IL-8. T cell antigen specific production of cytokinescan also be measured by intracellular cytokine staining withfluorescently labeled specific antibodies and flow cytometry or byELISPOT. Both CD4+ ad CD8+ T cell populations are studied.

Measurement of adjuvant activity at the cellular level is alsodetermined by immunophenotyping of surface markers of activation by flowcytometry. CD8 T cell antigen-specific responses are also evaluated byintracellular cytokine staining of perforin, cell surface markerexpression or proliferation assays including thymidine incorporation.

These experiments are designed to validate compound adjuvant activity indifferent combinations of prime-boost schemes and assess how the effectsof KIN1000 or related compounds on the innate immune antiviral programsshape the adaptive immune responses mounted to the antigen in thevaccine preparations.

Detailed immune response analyses of each compound as described aboveare conducted with each selected antigen to determine the immunecorrelates for that particular antigen(s) and compound formulation.These results guide the protection studies in which animals vaccinatedand boosted with combinations of select optimized compounds and desiredantigen(s) formulations from select infectious agents are laterchallenged with doses of infectious agent that are known to result indisease or death of the animal. Protection afforded by vaccination istypically measured by monitoring of clinical symptoms and survival.

A proof of concept experiment was performed. LEWIS female rats at 10-12weeks of age were primed with suspensions of antigen (ovalbumin, 0.2mg/Kg) and KIN1000 (1 mg/Kg) or KIN1148 (1 mg/mL) in phosphate salinebuffer (PBS) on day zero. Control animals received ovalbumin (OVA,InvivoGen Inc.) in PBS or OVA with poly I:C (0.1 mg/Kg, InvivoGen Inc.).Animals were boosted at weeks 2, and 8. Vaccines were deliveredsubcutaneously in the footpad and base of the tail for priming and onthe footpad and flank for the boosts (0.025 mL/site). Blood samples werecollected by tail bleed and processed to serum at 0, 1, 2, 4, 6, and 9weeks post priming. Titers of OVA specific antibodies were determined byELISA using anti IgM, anti IgG and anti IgG isotype specific antibodies.FIG. 10 shows IgG antibody levels relative to OVA alone vaccinatedcontrols in OVA+KIN1000 and OVA+KIN1148 vaccinated animals.

Measurement of cell-mediated adjuvant activity is also determined bydetermining delayed type hypersensitivity (DTH) to an antigen. In thesame proof of concept experiment, cellular responses were evaluated bydetermining the delayed type hypersensitivity reaction to challenge withOVA 2 weeks after the first boost. Animals were sedated with isofluraneand injected with PBS or OVA (0.02 mL of 1 m/gmL solution of OVA in PBS)in the pinna of the left and right ears, respectively. 24 hours laterthe difference in ear thickness was calculated. FIG. 11 shows measureddifference between right ear and left ear thickness.

Example 14 IRF-3 Activity for Structures with Amide Isostere LinkingGroups

Antiviral compounds of Formula III having amide isostere linkingstructures were synthesized and tested for IRF-3 activity, usingmethodology as reported herein. Antiviral compounds having linkinggroups L with the following structures were prepared and analyzed forIRF-3 activity. The structures and activity are reported in Table 7. Allstructures prepared displayed activity for IRF-3.

TABLE 7 IRF-3 Activity for Linker Analogs Struc- ture IRF-3 ID StructureActivity 1185

 1 1186

 8 1208

17 1209

23 1210

13 1218

11 1239

10

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that can varydepending upon the desired properties sought to be obtained by thepresent disclosure. At the very least, and not as an attempt to limitthe application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the disclosure are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the disclosure (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the disclosure and does not pose alimitation on the scope of the disclosure otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the disclosure.

Groupings of alternative elements or embodiments of the disclosuredisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group can be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this disclosure are described herein, includingthe best mode known to the inventors for carrying out the disclosure. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the disclosureto be practiced otherwise than specifically described herein.Accordingly, this disclosure includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by thedisclosure unless otherwise indicated herein or otherwise clearlycontradicted by context.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or and consisting essentially of language.When used in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the disclosure so claimed areinherently or expressly described and enabled herein.

It is to be understood that the embodiments of the disclosure disclosedherein are illustrative of the principles of the present disclosure.Other modifications that may be employed are within the scope of thedisclosure. Thus, by way of example, but not of limitation, alternativeconfigurations of the present disclosure may be utilized in accordancewith the teachings herein. Accordingly, the present disclosure is notlimited to that precisely as shown and described.

1-26. (canceled)
 27. A method of treating or preventing a viralinfection in a vertebrate comprising administering to the vertebrate apharmaceutical composition comprising a compound represented by aformula:

wherein a dashed line indicates the presence or absence of a pi bond; Aand B are each independently single or double covalent bonds;L is A-C(═R^(x))—NR⁵—B,A-SO₂—NR⁵—B,A-N R⁵—SO₂—B,A-CH(CF₃)—NR⁵—B,A-NR⁵—CH(CF₃)—B,

A-N R⁵—C(═R^(y))—NR⁵—B,A-CR²R³—R^(x)—B,A-O—CR²R³—B,A-S—CR²R³—B,A-C(R²)═C(R³)—B,

where m and n are independently an integer from 0-5 such that m+n≧1, R¹is R^(a), OR² or NR²R³; each R^(a) is independently H, hydrocarbyloptionally substituted by OR^(b), OCF₃, OCOR^(b), COOR^(b),CON^(b)R^(c), SR^(b), NR^(b)R^(c), NR^(b)COR^(c), SO₂NR^(b)R^(c), NO₂,F, Cl, Br, CN, heterocycloalkyl; aryl optionally substituted by OR^(b),OCF₃, OCOR^(b), COOR^(b), CON^(b)R^(c), SR^(b)NR^(b)R^(c),NR^(b)COR^(c), SO₂NR^(b)R^(c), NO₂, F, Cl, Br, CN, heterocycloalkyl; orheteroaryl optionally substituted by OR^(b), OCF₃, OCOR^(b), COOR^(b),CON^(b)R^(c), SR^(b), NR^(b)R^(c), NR^(b)COR^(c), SO₂NR^(b)R^(c), NO₂,F, Cl, Br, CN, heterocycloalkyl; R² and R³ are each independently R^(a),COR^(a), C(═O)OR^(a), or SO₂R^(a); Y⁵, Y⁶, Y⁷ and Y⁸ are eachindependently CR⁴, N, or O; R⁴ is R², OR^(a), NR²R³, SR^(a), SOR^(a),SO₂R^(a), SO₂NHR^(a), N(R⁵)COR^(a), halogen, trihalomethyl, CN, S═O, ornitro; each R⁵ is independently R^(a), COR^(a), SO₂R^(a), or is notpresent; W¹ and W² are each independently, O, S, NH, or CH₂; each R^(x)is independently O, S, CR²R³, or NR⁵; R^(y) is S, N—CN, or CHR⁴; R^(z)is R^(b), OR^(b), SR^(b), COR^(b), CO₂R^(b), OCOR^(b), NR^(b)R^(c),CON^(b)R^(c), NR^(b)COR^(c), SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I,or C₂₋₅ heterocyclyl; each R^(b) is independently H or C₁₋₃ hydrocarbyl,and each R^(c) is independently H or C₁₋₃ alkyl; and Z¹ and Z² are eachindependently C or N.
 28. The method of claim 27 wherein the compound isrepresented by a formula

wherein R¹⁰, R¹³, R¹⁴, R¹⁹, R²⁰, R²¹, and R²² are independently R^(b),OR^(b), SR^(b), COR^(b), CO₂R^(b), OCOR^(b), NR^(b)R^(c), CON^(b)R^(c),NR^(b)COR^(c), SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I, or C₂₋₅heterocyclyl; each R^(b) is independently H or C₁₋₃ hydrocarbyl, andeach R^(c) is independently H or C₁₋₃ alkyl.
 29. The method of claim 27,wherein R¹ is optionally substituted naphthyl or optionally substitutedphenyl.
 30. The method of claim 27, wherein the compound is furtherrepresented by a formula:


31. The method of claim of claim 27, wherein in the compound, R⁵ is H orC₁₋₃ alkyl.
 32. The method of claim 27, wherein the compound is furtherrepresented by a formula

wherein R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are independently R^(b), OR^(b),COR^(b), CO₂R^(b), OCOR^(b), NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, or I,wherein R^(b) and R^(c) are independently H or C₁₋₃ alkyl; and, R⁵ is Hor C₁₋₃ alkyl.
 33. The method of claim 27, wherein in the compound,R^(z) is CH₃.
 34. The method of claim 27, wherein in the compound, R¹³is Br.
 35. The method of claim 27, wherein the compound is furtherrepresented by a formula


36. The method of claim 27 wherein the viral infection is caused by avirus from one or more of the following families: Arenaviridae,Astroviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae,Closteroviridae, Comoviridae, Cystoviridae, Flaviviridae, Flexiviridae,Hepevirus, Leviviridae, Luteoviridae, Mononegavirales, Mosaic Viruses,Nidovirales, Nodaviridae, Orthomyxoviridae, Picobirnavirus,Picornaviridae, Potyviridae, Reoviridae, Retroviridae, Sequiviridae,Tenuivirus, Togaviridae, Tombusviridae, Totiviridae, Tymoviridae,Hepadnaviridae, Herpesviridae, Paramyxoviridae or Papillomaviridae 37.The method of claim 27 wherein the viral infection is influenza virus,Hepatitis C virus, West Nile virus, SARS-coronavirus, poliovirus,measles virus, Dengue virus, yellow fever virus, tick-borne encephalitisvirus, Japanese encephalitis virus, St. Louis encephalitis virus, MurrayValley virus, Powassan virus, Rocio virus, louping-ill virus, Banzivirus, Ilheus virus, Kokobera virus, Kunjin virus, Alfuy virus, bovinediarrhea virus, Kyasanur forest disease virus, respiratory syncytialvirus or human immunodeficiency virus (HIV).
 38. The method of claim 27,wherein the pharmaceutical composition is administered as an adjuvantfor a prophylactic or therapeutic vaccine.
 39. The method of claim 37wherein the method comprises vaccinating a vertebrate by additionallyadministering a vaccine against influenza virus, Hepatitis C virus, WestNile virus, SARS-coronavirus, poliovirus, measles virus, Dengue virus,yellow fever virus, tick-borne encephalitis virus, Japanese encephalitisvirus, St. Louis encephalitis virus, Murray Valley virus, Powassanvirus, Rocio virus, louping-ill virus, Banzi virus, Ilheus virus,Kokobera virus, Kunjin virus, Alfuy virus, bovine diarrhea virus,Kyasanur forest disease virus, respiratory syncytial virus or humanimmunodeficiency virus (HIV).
 40. A method of modulating the innateimmune response in a eukaryotic cell, comprising administering to theeukaryotic cell a compound represented by a formula:

wherein a dashed line indicates the presence or absence of a pi bond; Aand B are each independently single or double covalent bonds;L is A-C(═R^(x))—NR⁵—B, A-SO₂—NR⁵—B,A-NR⁵—SO₂—B,A-CH(CF₃)—NR⁵—B,A-NR⁵—CH(CF₃)—B.

A-NR⁵—C(═R^(y))—NR⁵—B,A-CR²R³—R^(x)—B,A-O—CR²R³—B,A-S—CR²R³—B,A-C(R²)═C(R³)—B,

where m and n are independently an integer from 0-5 such that m+n≧1, R¹is R^(a), OR² or NR²R³; each R^(a) is independently H, hydrocarbyloptionally substituted by OR^(b), OCF₃, OCOR^(b), COOR^(b),CON^(b)R^(c), SR^(b), NR^(b)R^(c), NR^(b)COR^(c), SO₂NR^(b)R^(c), NO₂,F, Cl, Br, CN, heterocycloalkyl; aryl optionally substituted by OR^(b),OCF₃, OCOR^(b), COOR^(b), CON^(b)R^(c), SR^(b), NR^(b)R^(c),NR^(b)COR^(c), SO₂NR^(b)R^(c), NO₂, F, Cl, Br, CN, heterocycloalkyl; orheteroaryl optionally substituted by OR^(b), OCOR^(b), COOR^(b),CON^(b)R^(c), SR^(b), NR^(b)R^(c), NR^(b)COR^(c), SO₂NR^(b)R^(c), NO₂,F, Cl, Br, CN, heterocycloalkyl; R² and R³ are each independently R^(a),COR^(a), C(═O)OR^(a), or SO₂R^(a); Y⁵, Y⁶, Y⁷, and Y⁸ are eachindependently CR⁴, N, or O; R⁴ is R², OR^(a), NR²R³, SR^(a), SOR^(a),SO₂R^(a), SO₂NHR^(a), N(R⁵)COR^(a), halogen, trihalomethyl, CN, S═O, ornitro; each R⁵ is independently R^(a), COR^(a), SO²R^(a), or is notpresent; W¹ and W² are each independently, O, S, NH, or CH₂; each R^(x)is independently O, S, CR²R³, or NR⁵; R^(y) is S, N—CN, or CHR⁴; R^(z)is R^(b), OR^(b), SR^(b), COR^(b), CO₂R^(b), OCOR^(b), NR^(b)R^(c),CON^(b)R^(c), NR^(b)COR^(c), SO₂NR^(b)R^(c), CF₃, CN, NO₂, F, Cl, Br, I,or C₂₋₅ heterocyclyl; each R^(b) is independently H or C₁₋₃ hydrocarbyl,and each R^(c) is independently H or C₁₋₃ alkyl; and Z¹ and Z² are eachindependently C or N.
 41. The method of claim 40, wherein the eukaryoticcell is in viva
 42. The method of claim 40, wherein the eukaryotic cellis in vitro.